Biodegradation of textile dyes by immobilized laccase ...

International Biodeterioration & Biodegradation 90 (2014) 71e78

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Biodegradation of textile dyes by immobilized laccase from Coriolopsis gallica into Ca-alginate beads Dalel Daâssi a, Susana Rodríguez-Couto b, c, Moncef Nasri a, Tahar Mechichi a, * a Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, Laboratoire de Génie Enzymatique et de Microbiologie, Route de Soukra Km 4,5 BP «1173», 3038 Sfax, Tunisia b CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain c IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 January 2014 Received in revised form 16 February 2014 Accepted 17 February 2014 Available online

Synthetic dyes are extensively used in a number of industries, such as textile dyeing. Due to their low biodegradability, they cause serious environmental pollution. Thus, in the present paper a partiallypurified acid fungal laccase from the white-rot basidiomycete Coriolopsis gallica was entrapped into calcium alginate beads and applied to the decolorization of different synthetic dyes. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration and the ratio enzyme/alginate (E/A) on the loading efficiency and immobilization yield were investigated. The optimal conditions for C. gallica laccase immobilization into Ca-alginate beads were 2% (w/v) sodium alginate, 2% (w/v) CaCl2, and 1:4 E/A (v/v). It was also found that laccase stability to pH and temperature increased after immobilization. Both the free and immobilized laccase alone showed a high efficiency to decolorize the anthraquinone dye Remazol Brilliant Blue R (RBBR) while a low decolorization yield was observed for the diazo dyes Reactive Black 5 (RB5) and Bismark Brown R (BBR) and the metal textile dye Lanaset Grey G (LG). The addition of the redox mediator 1-hydroxybenzotriazole (HBT) to the decolorization reaction increased significantly dye removal. The immobilized laccase retained 70% of its activity after four successive decolorization cycles except for BBR (51.2%). The results obtained showed that the immobilized laccase from C. gallica has potential for its application in dyestuff treatment. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Fungal laccase System laccase-HBT Stability Decolorization Reusability

1. Introduction Fungal laccases have been subject of increased research in the last decades, since their wide substrate specificity for the reducing substrates make these enzymes particularly useful for a wide variety of industrial applications (Rodríguez-Couto and Toca Herrera, 2006; Kunamneni et al., 2008), including pulp bleaching in the paper industry (Moldes et al., 2010), decolorization of textile dyes (Khlifi et al., 2009; Daâssi et al., 2012), biofuel cells (Li et al., 2011), biosensors (Ardhaoui et al., 2013), green chemistry (Witayakran and Ragauskas, 2009) and bioremediation and detoxification of environmental pollutants (Lloret et al., 2012). Among the above-mentioned applications, the use of laccases as biocatalysts in the treatment of textile effluents seems a promising approach (Enayatzamir et al., 2009; Benzina et al., 2013). Indeed,

* Corresponding author. Tel.: þ216 74 274 088; fax: þ216 74 275 595. E-mail addresses: [email protected], [email protected] (T. Mechichi). http://dx.doi.org/10.1016/j.ibiod.2014.02.006 0964-8305/Ó 2014 Elsevier Ltd. All rights reserved.

more than 10000 different types of dyes and about 80,000 tons of dyes are produced commercially worldwide per year and between 5e10% are incorporated into wastewater by different ways (Hessel et al., 2007). Laccase has been reported to decolorize several synthetic dyes (Michniewicz et al., 2008; Daâssi et al., 2012). However, the soluble laccase used in those applications showed some disadvantages such as stability lost and non-reusability, making the laccase treatment expensive. Thus, different approaches were used to reduce the production cost of laccases in order to make their application more economical. Among such approaches laccase immobilization allows its reuse and improves its stability (Betancor et al., 2013). Thus, by mimicking the natural mode of occurrence in living cells, where most of the enzymes are attached to cellular membranes, immobilization stabilizes the structure of enzymes, and, hence, their activities (Addorisio et al., 2013). Furthermore, immobilization can also improve enzyme performance under the optimal conditions of the different industrial process (Spinelli et al., 2013). In addition, immobilization also makes product separation easier, thereby, permitting continuous processes and, thus,

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D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e78

preventing the loss of protein or activity in subsequent process steps (Polizzi et al., 2007). Laccase immobilization has been extensively studied using different methods and supports (Sanlıer et al., 2013; Li et al., 2013). Among the different immobilization methods, entrapment may be a good choice as it is a mild process and causes relatively little damage to the native structure of the enzyme (Duran et al., 2002). Entrapment in calcium alginate gel offers many advantages due to its simplicity, eco-friendly nature and cost-effectiveness (Rao et al., 2009). Alginate supports are usually made by cross linking the carboxyl group of the a-L-guluronic acid with a solution of a cationic cross linker such as calcium chloride, barium chloride or poly(L-lysine) (Draget et al., 1997). Several studies have been recently published about the application of immobilized laccases (Osma et al., 2010). In the present study, laccase was immobilized into alginate beads and the conditions for immobilization and characterization of the free and immobilized enzyme were investigated. The reusability and stability (pH stability, thermal stability and storage stability) of immobilized laccase were also studied and compared with those of the free enzyme. In addition, the capability of both free and immobilized laccase to decolorize different textile dyes was assessed. 2. Materials and methods 2.1. Chemicals Sodium alginate and calcium chloride dihydrate (CaCl2$2H2O), were obtained from Fluka, Norway. The synthetic dyes Remazol Brilliant Blue R (RBBR, dye content 50%), Reactive Black 5 (RB5, dye content 55%) and Bismark Brown R (BBR, dye content 55%) were purchased from SigmaeAldrich, USA. The complex metal dye Lanaset Grey G (LG, dye content not available) was provided by DyStar, Portugal. All other chemicals were of analytical grade and used without further purification. 2.2. Microorganism The strain used in this study was newly isolated from decayed acacia wood in the Northwest of Tunisia. The fungal strain was identified as Coriolopsis gallica based on morphological and molecular methods. The ITS sequence was deposited in Genebank under accession number KJ412304. C. gallica strain BS54 was maintained on 2% (w/v) malt extract agar (MEA) and conserved as growing culture in the culture collection of our laboratory (Laboratory of Enzyme Engineering and Microbiology, University of Sfax). 2.3. Culture conditions C. gallica KJ412304 was cultured in semi-solid-state fermentation conditions in 250-mL cotton-plugged Erlenmeyer flasks containing 5.0 g of sawdust. The substrate was hydrated with 15 mL of minimum medium (MM) adjusted by 25 mM acetate buffer (pH 5.0). This MM contained (g L1): glucose, 5; casein peptone, 6; KH2PO4, 0.025; MgSO4$7H2O, 0.25; KCl, 0.5. Inoculation was carried out directly in the Erlenmeyer flasks. Six plugs (diameter, 3 mm), from a 5-day growing fungus on malt extract agar (MEA) plates, per Erlenmeyer were used as inoculum. The cultures were supplemented with CuSO4 as laccase-inducer (solution sterilized separately, 60 mM) at the beginning of the cultivation. The Erlenmeyer flasks were incubated statically for 12 days under an air atmosphere at 30 C and 90% humidity, to avoid evaporation, in complete darkness.

At the end of cultivation (12 days) the flask contents were extracted with sodium acetate buffer (pH 5.0, 25 mM) under shaking for 1 h, filtered and centrifuged at 7000 rpm for 20 min at 4 C. The supernatant was collected and used as enzyme source for quantification of laccase enzyme. 2.4. Partial purification of C. gallica laccase The culture broth was concentrated by ultrafiltration (Filtron, 3 kDa cut-off). This concentrate was loaded onto a 5-mL HiTrap QFF column (GE Healthcare) pre-equilibrated with 25 mM sodium acetate pH 5.5 and the retained proteins were eluted with a 0e 250 mM NaCl gradient (140 min, 1 mL min1). The laccase active fractions were pooled, concentrated and dialyzed against the above buffer, pH 5.0, in a stirred cell apparatus (Amicon, 3 kDa cut-off). 2.5. Immobilization of laccase In order to preserve enzyme activity and to achieve high immobilization efficiency, the gelling agent (sodium alginate and calcium chloride solution) and the quantity of enzyme introduced should be studied and optimized. For this purpose varying concentrations of sodium alginate (1.0, 1.5, 2.0, 2.5, 3.0 and 4.0% w/v) and calcium chloride (1e4% w/v) were used during immobilization of C. gallica laccase to achieve 100% immobilization yield. Alginate solution (1.0, 1.5, 2.0, 2.5, 3.0 and 4.0% (w/v)) was prepared by dissolving sodium alginate in deionized water containing a certain amount of laccase (200e800 mg L1). Then different ratios of enzyme/alginate (E/A) (v/v) were mixed under shaking (Baysal, 2007). The mixture was dropped by means of a syringe into a CaCl2 solution (1e4% w/v) under shaking. After 1 h the beads (about 3e4 mm in diameter) were collected from the solution and washed with CaCl2 0.5% (w/v) twice and then washed three times with deionized water. The filtered hardening solutions and the two washings were collected for loading efficiency (Eq. (1)) and immobilization yields determination (Eq. (2)). Loading efficiency and immobilized yield were defined as the percentage of total enzyme entrapped and the specific activity ratio of entrapped laccase to free laccase, respectively.

Loading efficiency ð%Þ ¼

h

Ci Vi Cf Vf

. i Ci 100

(1)

Where Ci is the initial protein concentration, Vi the initial volume of enzyme solution, Cf the protein concentration in the total filtrate and Vf the total volume of the filtrate.

Immobilization ð%Þ ¼ ððAi Awash Þ=Ai Þ 100

(2)

Where, Ai is the initial activity of the free enzyme introduced into the mixture of Ca-alginate solution assayed using ABTS [2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate and Awash is the laccase activity detected in the curing solution and the two washing solutions assayed using the same substrate. All the immobilized laccase beads were kept in distilled water at 4 C until further use. 2.6. Enzyme assay and protein estimation The activity of the free and immobilized laccase was assayed using 1 mM ABTS as a substrate. For the free enzyme the reaction was initiated by adding 0.3 mL of ABTS to a mixture consisting of 3 mL of 100 mM succinic acid buffer (pH 5.0) and the enzyme (Erden et al., 2009). The molar extinction coefficient of ABTS is 36000 M1 cm1. The increase in absorbance was recorded at


420 nm for 1 min in a UV-vis spectrophotometer (Perkin Elmer). For the immobilized enzyme 10 beads of immobilized laccase, 2.7 mL of 100 mM succinic acid buffer (pH 5.0) and 0.3 mL of ABTS were incubated under shaking at 100 rpm for 5 min. Oxidation of ABTS was followed by an absorbance increase at 420 nm. One activity unit (U) of laccase was defined as the amount of enzyme required to catalyze 1 mmoL mL1 of substrate per minute. All the assays were carried out in triplicate. Protein estimation of free laccase was performed using the method of Bradford using the commercial reagent Bio-Rad (Sigma Chemical, St. Louis, USA) and bovine serum albumin (BSA) as a standard (Bradford, 1976). For the immobilized laccase, the amount of protein in the supernatant solution after immobilization was determined using the Bradford method. Bound proteins were determined as the difference between the initial and the residual protein concentrations. 2.7. Characterization of the immobilized laccase 2.7.1. Stability tests The effect of pH on laccase immobilized beads was compared and studied by incubating the samples in 100 mM succinic acid buffer ranging from pH 2.0 to 9.0 for 24 h. The residual activity was estimated at the end of the incubation period for all the different pH ranges as described in the Section 2.6. Thermal stability was assayed by incubating the laccase immobilized beads and the free laccase simultaneously at 55 C for 210 min. The residual activity was measured as described in the Section 2.6. Storage stability experiment was performed to determine the stabilities of free and immobilized laccase. For storage stability measurements, immobilized laccase was kept at 4 C without buffer. The activity of immobilized laccase was followed for 20 days and determined by the laccase activity assay procedure reported in the Section 2.6. Then, the immobilized laccase was reused each 2 days and the variation of activity was measured with respect to the initial one. After each assay, laccase immobilized beads were washed with buffer and stored at 4 C for further uses.

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beads. Seven decolorization cycles of 24 h each were performed for each of the different tested dyes. After each cycle, the beads were removed and washed with 50 mM Tris HCl buffer (pH 7.2) and the solution replaced with fresh dye solution. The activity of freshly prepared beads in the first run was defined as 100%. The reusability study was performed in triplicate. 2.8. Decolorization studies of immobilized laccase on different synthetic textile dyes Four synthetic dyes, Remazol Brilliant Blue R (RBBR), Reactive Black 5 (RB5), Bismark Brown R (BBR) and Lanaset Grey G (LG: complex metal dye) were selected as model dyes to study the decolorization ability of the C. gallica laccase immobilized into Ca-alginate beads (Table 1). Stock solutions of the dyes (0.1% w/v in water) were stored in the dark at room temperature. Experiments were performed using 50-mL disposable flasks in a 5-mL final reaction volume. The reaction mixture, containing 10% (v/v) beads/100 mM succinic buffer pH 5.0 and 1 mM of HBT (in laccase mediator systems), was incubated statically in the dark at 30 C. Dye concentrations were selected in order to obtain around 1.5 absorbance units at the maximum wavelength of each dye in the visible spectrum (75 mg L1 for RBBR, 66.7 mg L1 for RB5, 36.0 mg L1 for BBR and 100.0 mg L1 for LG). A control reaction with Ca-alginate beads without laccase was prepared under the same conditions to detect possible removal of color due to dye adsorption onto the alginate beads. All the experiments were performed in duplicate. Dye concentrations were spectrophotometrically (Shimadzu UV 1650 PC) measured from 400 to 800 nm and calculated by measuring the area under the plot. Decolorization was calculated by the following equation:

Decolorization ð%Þ ¼

. Absinitial 100; Absinitial Absfinal

Where Absinitial was the area under the curve from 300 to 800 nm at the initial time and Absfinal was the area under the curve from 300 to 800 nm at a particular time. 2.9. Data analysis

2.7.2. Reusability Ca-alginate beads were used several times for the different dye decolorization reactions to test the reusability of laccase entrapped

Mean and standard deviation (SD) of the results from at least three independent experiments were calculated using Microsoft

Table 1 Characteristics of the synthetic dyes used. Dye

CI number

CI name

Class

lmax

Remazol Brilliant Blue R (RBBR)

61200

Reactive Blue 19

Anthraquinonic

592

Reactive Black 5 (RB5)

20505

Reactive Black 5

Diazo

597

Bismark Brown R (BBR)

21010

Basic Brown 4

Diazo

468

Lanaset Grey G (LG)

e

e

Complex metal dye

579

CI: color index.

Structure

Not disclosed

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Table 2 -Purification of the extracellular laccase from Coriolopsis gallica. Purification steps

Volume (mL)

Laccase activity (U)

Protein content (mg)

Specific activity (U/mg)

Purification fold

Yield (%)

Culture extract Ultrafiltration HiTrap QFF Ultrafiltration

250 40 200 40

1450 1080 980 880

105 31.5 16.4 12.8

13.8 34.2 59.7 68.7

1 2.4 4.3 4.9

100 74.4 67.5 60.6

Excel-software (Microsoft). Readings were considered significant when p was 0.05. 3. Results and discussion 3.1. Laccase production and purification Laccase was produced from a locally-isolated strain of the white-rot-fungus C. gallica KJ412304 grown on sawdust as described in 2.3. The cell-free extracellular liquid produced was subjected to partial purification by ultrafiltration and column chromatography as summarized in Table 2. 3.2. Laccase immobilization into alginate beads The gelation of alginate could be initiated by mixing sodium alginate and calcium chloride solution. However, the preparation of beads with rigidity and proper permeability for enzyme gel entrapment is based on both, the concentration of sodium alginate

and the ability of calcium ions to cross link with sodium alginate, and also the ratio Enzyme/Alginate (E/A) (Phetsom et al., 2009). The following parameters depict the immobilization efficiency of the purified laccase from C. gallica (Fig. 1aec). The loading efficiency (percent of total enzyme entrapped) and immobilization yield (specific activity ratio of entrapped laccase to free laccase) are defined as described in Eqs. (1) and (2), respectively. In Fig. 1a, the effect of various alginate concentrations (from 1 to 4% (w/v)), maintaining the calcium chloride concentration at 2% (w/ v) and the ratio E/A at 1:4 is shown. From the values in the above mentioned figure, it is shown that the maximum entrapped laccase occurred at 0.5 and 1% (w/v) sodium alginate concentration owing to the larger pore size of the less tightly crossed linked fragile Caalginate beads. However, there was a gradual decrease in the immobilization efficiency with 3% and 4% sodium alginate. This may be attributed to the pore size of the beads and the degree of cross-linking of the gelling agent. Riaz et al. (2009) found that the lower concentration of sodium alginate solution leads to the greater pore size of the beads resulting in increased leakage of the enzyme from the beads. Similarly, the higher the concentration of sodium alginate, the smaller the pore size of the beads leading to lower immobilization efficiency. In addition, an increase in alginate increases the viscosity of the solution making enzyme encapsulation cumbersome (Geethanjali and Subash, 2013). From the Fig. 1a, it is observed that sodium alginate at a concentration of 2.5% (w/v) registered the highest entrapped laccase activity with the loading efficiency value being 83 0.5%. This is in accordance with the results of Geethanjali and Subash (2013), who stated that sodium alginate ranging from 2 to 3%, was suitable for immobilization of protease.

Fig. 1. (a) Effects of alginate concentration. Immobilization conditions: Laccase solution (0.2e1 mg mL1), CaCl2 solution (1% w/v), Enzyme/Alginate ratio (1:4); (b) Effects of CaCl2 concentration. Immobilization conditions: Laccase solution (0.5 mg/mL), alginate solution (2% w/v). Loading efficiency (bar) and immobilization yield (A); (c) Effect of Enzyme/ Alginate ratio (v:v). Immobilization conditions: Laccase solution (0.5 mg mL1), alginate (2% w/v), CaCl2 (2% w/v).


Furthermore, calcium chloride is used as a cross linking agent, and its concentration affects the activity and the stability of immobilized enzyme. Fig. 1b shows that an increase in the calcium chloride concentration from 1.5 to 3% (w/v) at 2% (w/v) alginate concentration had little effect on the loading efficiency (77.3 1.1%, 82.2 2.4%, respectively). Former studies showed that the diffusion of high molecular weight substances from the Ca-alginate beads into the bulk solution was little affected by increases in the CaCl2 concentration (1e2% w/v), but it was considerably limited by increases in alginate concentration (2e4% w/v) (Tanaka et al., 1984). Therefore, alginate concentration plays a key role for enzyme entrapment into Ca-alginate beads. Moreover, the effect of ratio (E/A) on the loading efficiency and immobilization yield is shown in Fig. 1c. Since the beads prepared in the present work were very small, their loading efficiency might be relatively limited which would cause a decrease in the immobilization yield at a higher E/A ratio (1:8). The finding that the ratio (E/A) of 1:4 was found to be the optimum for the immobilization of purified laccase from C. gallica can be contrasted with the reports of Lu et al. (2007), who reported that immobilization of enzyme by alginate-chitosan microcapsules using a ratio (E/A) of 1/ 8 was found to be the best for laccase entrapment. The optimal conditions were 2% (w/v) sodium alginate, 2% (w/v) CaCl2 and 1:4 E/A ratio. Under such conditions, the loading efficiency and immobilized yield of the immobilized laccase were 88.1 2.4% and 93.3 1.1%, respectively. 3.3. Characterization of the free and immobilized laccase 3.3.1. Stability tests The thermal stability of the free and immobilized C. gallica laccase was investigated at 55 C for different incubation times. This temperature was selected because it is the temperature normally used in the textile industry for dyeing. The data from the Fig. 2, show that the immobilized and free laccase maintained 91.2 0.7% and 25.6 1.3% of their initial activities, respectively, after 90 min of incubation at 55 C. The residual activity at 210 min of incubation was about 67 0.8% for the immobilized laccase and 3% for the free enzyme. This is in accordance with the results of Reyes et al. (1999), who found that an immobilized laccase from C. gallica UAMH8260 on activated agarose showed higher thermal stability at 70 C than the free enzyme.

Fig. 2. Thermostability of immobilized and free laccase at 55 C.

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The pH stability curve of immobilized and free laccase is depicted in Fig. 3. From the values in the above mentioned figure, it can be deduced that compared to the free laccase, the immobilized laccase stability in acidic pH values was higher (about 30%). Also, the stability of the immobilized laccase in the pH range 7.0e9.0 was about 20% higher than the free enzyme. Generally, free enzymes can lose their activities quickly (Cevik et al., 2011). Hence, it is advisable to immobilized enzymes. Storage stability is one of the most important parameters to be considered in biocatalyst immobilization. Fig. 4 indicates that, at the end of the 20 days of storage, the free laccase and immobilized laccase retained about 22.4 1.8% and 82.7 0.9% of their initial activities, respectively. Similar findings were observed by Huang et al. (2006), who reported that after storage at 4 C for one month, the activity of Pycnoporus sanguineus laccase immobilized on copper tetra-aminophthalocyanine-Fe3O4 nanoparticle composite was 85% of its initial activity, while that of the free laccase was only 30%. 3.3.2. Decolorization of different synthetic dyes by free and immobilized laccase The dye-decolorizing potential of immobilized and free laccase from C. gallica was demonstrated for different textile dyes belonging to 3 dye families: an anthraquinone dye (RBBR), diazo dyes (RB-5 and BBR) and a complex metal dye (LG) (Fig. 5aed). From data in Fig. 5a, it can be observed that RBBR was rapidly decolorized by both the free and immobilized laccase alone, compared to LG, RB5 and BBR (Fig. 5bed). For the diazo and the complex metal dye, the maximum decolorization yields obtained were not higher than 50% within 24 h of incubation without redox mediators, whereas they were more efficient in the presence of HBT (Fig. 5bed). In the control, Ca-alginate beads were able to remove about 12% of initial RBBR dye within 90 min. While by free enzyme, this anthraquinone dye was decolorized to 74.6% and increased up to 90.3% using immobilized laccase after 90 min of treatment (Fig. 5a). Furthermore, the decolorization levels of RBBR did not improve by adding 1 mM of HBT (Fig. 5a). The above results are supported by Mechichi et al. (2006), who emphasized no effect of the redox mediator HBT at concentrations between 0.125 and 2.5 mM on the decolorization of RBBR. Other results also reported that the oxidation of RBBR was easily carried out by laccase alone (Kunamneni et al., 2008; Khlifi et al., 2009). Decolorization time-course of BBR by immobilized and free laccase is depicted in Fig. 5b. The results show a high percentage of dye adsorption onto the beads (34.6 0.6%) within 24 h compared

Fig. 3. Stability of the immobilized laccase to pH variations.

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Fig. 4. Storage stability of the free and immobilized laccase.

to the other dyes studied (Fig. 5a, d). In the case of entrapped and free enzyme, this azo dye was decolorized to 52.8 1.7% and 47.1 1.1%, respectively, within 24 h. Thus, the immobilized laccase was not able to degrade the dye adsorbed onto the beads and dye decolorization was mainly due to adsorption (Fig. 5b). The addition of HBT did no enhance significantly the color removal rates. These results are in agreement with the study performed by Enayatzamir et al. (2010), who stated that the dye BBR was resistant to biodegradation by the white-rot fungus P. chrysosporium immobilized into alginate beads and dye removal was mainly due to dye adsorption onto the alginate beads. The higher affinity of BBR for adsorption onto alginate beads might be related to the different ionisation of this dye, since it is a cationic dye whereas the other tested dyes are anionic (Enayatzamir et al., 2010).

Fig. 5c illustrates the decolorization yields of the dye LG by laccase and laccase-mediator system. LG decolorization performed with immobilized laccase showed a removal percentage of 49 0.4% in 24 h and the free enzyme of 30.5 3.4% for the same period of time. In the presence of HBT as a redox mediator maximum decolorization of 86.5 1.4% and 72.2 0.2% was achieved after 24 h with the immobilized and the free enzyme, respectively. LG is a recalcitrant commercial mixture of metalcomplex dyes that contains chromium (Crþ3) and cobalt (Coþ2) (Blánquez et al., 2004). Most of the previous studies on LG decolorization involved whole fungal cultures (Blánquez et al., 2004, 2007; Gabarrell et al., 2012; Daâssi et al., 2013) but there are no studies with laccase or laccase-mediator system. In the presence of HBT (1 mM) the immobilized enzyme exhibited efficient LG decolorization (86.5%) after 24 h. In the case of the azo dye RB5, the immobilized enzyme showed around 31% decolorization in 8 h and 24 h of incubation without HBT. However, the addition of 1 mM HBT enhanced the decolorization nearly to 1.3-fold by the immobilized enzyme, and, thus, a maximum decolorization of 78.2 0.7% was observed within 8 h (Fig. 5d). Kunamneni et al. (2008), found a similar result in the decolorization of RB5 by a laccase from Myceliophthora thermophila. Generally, the mechanism of dye removal using immobilized enzyme or cells may be due to either enzymatic biodegradation or bioaccumulation/biosorption of the dye onto alginate beads (Rodríguez-Couto, 2009; Daâssi et al., 2013). In this study, to detect the possible removal of color due to dye adsorption onto the alginate beads, a control reaction with Caalginate beads without laccase was prepared. Dye removal mechanism can also be judged clearly by inspecting the color of the beads up to the treatment dye process. It was observed that the alginate beads became colored especially after contacting with BBR and LG. From the Fig. 5, it is clearly understood that the BBR and LG removal mechanism is essentially biosorption of the dye onto

Fig. 5. Decolorization time-course of synthetic dyes (a) RBBR (b) BBR (c) LG (d) RB5 by immobilized or free laccase in the presence and absence of redox mediator (HBT, 1 mM).


alginate beads, with percentages of 34% and 24%, respectively, compared to the other dyes studied. Ca-alginate beads were able to remove only 12% and 17% of RBBR and RB5 dye, respectively. Thus, the predominant mechanism involved in RBBR and RB5 removal was laccase biodegradation. 3.3.3. Reusability In this stage of research, it was investigated whether the alginate-immobilized C. gallica laccase could be successfully reused after storage at 4 C. Reusability of immobilized enzymes in the biodegradation process exhibits the most important aspect for industrial applications, since it decreases the cost of the process. Thus, the reusability of the immobilized laccase in seven successive batches of 24 h each was investigated. The relative decolorization rates are depicted in Fig. 6. From data in the above figure, it can be detected that after the 4th cycle, the relative decolorization values for tested dyes were found to be more than 70% except BBR (51.2%) and showed lower yields at the end of the seven cycles. The gradual decrease in decolorization in the subsequent cycles has been explained differently in the literature. This may be related with enzyme inactivation. Indeed, upon repeated uses, the blocking of some pores of beads by substrate or product may take place. This restriction may cause a decrease in the efficient activity of C. gallica laccase entrapped into the gel after successive decolorization cycles. However, Anwar et al. (2009) emphasized that the decrease in activity occurred on further reuse may be due to the leakage of enzyme from alginate beads during washing at the end of each cycle. In the literature, there are several articles reporting the successful reuse of various immobilized laccase systems. Thus, a laccase from Panus conchatus immobilized on activated polyvinyl alcohol retained 60% of its activity after ten batch uses and more than 50% after 17 batch uses (Yinghui et al., 2002) and a laccase from P. sanguineus immobilized on copper tetraaminophthalocyanine (CuTAPc)-Fe3O4 magnetic nano-composite retained 80% of its initial activity after 5 bath uses (Xiao et al., 2006). 4. Conclusions From the current study it can be concluded that the immobilization of laccase into Ca-alginate beads improved its thermal and storage stabilities. Thus, the immobilized and free laccase maintained 91.2% and 25.6% of their initial activities, respectively, after 90 min of incubation at 55 C. Also, at the end of 20 days of storage,

Fig. 6. Reusability of immobilized laccase in the reaction condition of decolorization of: LG (>) BBR (,) RBBR (6) RB5 (B). Data were mean values SD. 7 decolorization cycles, 24 h each.

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