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International Biodeterioration & Biodegradation 90 (2014) 71e78

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International Biodeterioration & Biodegradation

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Biodegradation of textile dyes by immobilized laccase from Coriolopsisgallica into Ca-alginate beads

Dalel Daâssi a, Susana Rodríguez-Couto b,c, Moncef Nasri a, Tahar Mechichi a,*aUniversité de Sfax, Ecole Nationale d’Ingénieurs de Sfax, Laboratoire de Génie Enzymatique et de Microbiologie, Route de Soukra Km 4,5 BP «1173», 3038Sfax, TunisiabCEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spainc IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spain

a r t i c l e i n f o

Article history:Received 3 January 2014Received in revised form16 February 2014Accepted 17 February 2014Available online 12 March 2014

Keywords:Fungal laccaseSystem laccase-HBTStabilityDecolorizationReusability

* Corresponding author. Tel.: þ216 74 274 088; faxE-mail addresses: tahar.mechichi@enis.rnu.tn, tahar

http://dx.doi.org/10.1016/j.ibiod.2014.02.0060964-8305/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Synthetic dyes are extensively used in a number of industries, such as textile dyeing. Due to their lowbiodegradability, they cause serious environmental pollution. Thus, in the present paper a partially-purified acid fungal laccase from the white-rot basidiomycete Coriolopsis gallica was entrapped intocalcium alginate beads and applied to the decolorization of different synthetic dyes. Effects of immo-bilization 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 forC. 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 afterimmobilization.

Both the free and immobilized laccase alone showed a high efficiency to decolorize the anthraquinonedye Remazol Brilliant Blue R (RBBR) while a low decolorization yield was observed for the diazo dyesReactive Black 5 (RB5) and Bismark Brown R (BBR) and the metal textile dye Lanaset Grey G (LG). Theaddition of the redox mediator 1-hydroxybenzotriazole (HBT) to the decolorization reaction increasedsignificantly dye removal. The immobilized laccase retained 70% of its activity after four successivedecolorization cycles except for BBR (51.2%). The results obtained showed that the immobilized laccasefrom C. gallica has potential for its application in dyestuff treatment.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Fungal laccases have been subject of increased research in thelast decades, since their wide substrate specificity for the reducingsubstrates make these enzymes particularly useful for a wide va-riety of industrial applications (Rodríguez-Couto and Toca Herrera,2006; Kunamneni et al., 2008), including pulp bleaching in thepaper 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 (Witayakranand Ragauskas, 2009) and bioremediation and detoxification ofenvironmental pollutants (Lloret et al., 2012).

Among the above-mentioned applications, the use of laccases asbiocatalysts in the treatment of textile effluents seems a promisingapproach (Enayatzamir et al., 2009; Benzina et al., 2013). Indeed,

: þ216 74 275 595.98@yahoo.com (T. Mechichi).

more than 10000 different types of dyes and about 80,000 tons ofdyes are produced commercially worldwide per year and between5e10% are incorporated into wastewater by different ways (Hesselet al., 2007). Laccase has been reported to decolorize several syn-thetic dyes (Michniewicz et al., 2008; Daâssi et al., 2012). However,the soluble laccase used in those applications showed some dis-advantages such as stability lost and non-reusability, making thelaccase treatment expensive. Thus, different approaches were usedto reduce the production cost of laccases in order to make theirapplication more economical. Among such approaches laccaseimmobilization allows its reuse and improves its stability (Betancoret al., 2013). Thus, by mimicking the natural mode of occurrence inliving cells, where most of the enzymes are attached to cellularmembranes, immobilization stabilizes the structure of enzymes,and, hence, their activities (Addorisio et al., 2013). Furthermore,immobilization can also improve enzyme performance under theoptimal conditions of the different industrial process (Spinelli et al.,2013). In addition, immobilization also makes product separationeasier, thereby, permitting continuous processes and, thus,

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preventing the loss of protein or activity in subsequent processsteps (Polizzi et al., 2007). Laccase immobilization has beenextensively studied using different methods and supports (Sanlıeret al., 2013; Li et al., 2013). Among the different immobilizationmethods, entrapment may be a good choice as it is a mild processand causes relatively little damage to the native structure of theenzyme (Duran et al., 2002).

Entrapment in calcium alginate gel offers many advantages dueto its simplicity, eco-friendly nature and cost-effectiveness (Raoet al., 2009). Alginate supports are usually made by cross linkingthe carboxyl group of the a-L-guluronic acid with a solution of acationic cross linker such as calcium chloride, barium chloride orpoly(L-lysine) (Draget et al., 1997). Several studies have beenrecently published about the application of immobilized laccases(Osma et al., 2010).

In the present study, laccase was immobilized into alginatebeads and the conditions for immobilization and characterizationof the free and immobilized enzyme were investigated. The reus-ability and stability (pH stability, thermal stability and storagestability) of immobilized laccase were also studied and comparedwith those of the free enzyme. In addition, the capability of bothfree and immobilized laccase to decolorize different textile dyeswas assessed.

2. Materials and methods

2.1. Chemicals

Sodium alginate and calcium chloride dihydrate (CaCl2$2H2O),were obtained from Fluka, Norway. The synthetic dyes RemazolBrilliant Blue R (RBBR, dye content 50%), Reactive Black 5 (RB5, dyecontent 55%) and Bismark Brown R (BBR, dye content 55%) werepurchased from SigmaeAldrich, USA. The complex metal dyeLanaset Grey G (LG, dye content not available) was provided byDyStar, Portugal. All other chemicals were of analytical grade andused without further purification.

2.2. Microorganism

The strain used in this study was newly isolated from decayedacacia wood in the Northwest of Tunisia. The fungal strain wasidentified as Coriolopsis gallica based on morphological and mo-lecular methods. The ITS sequence was deposited in Genebankunder accession number KJ412304. C. gallica strain BS54 wasmaintained on 2% (w/v) malt extract agar (MEA) and conserved asgrowing culture in the culture collection of our laboratory (Labo-ratory of Enzyme Engineering andMicrobiology, University of Sfax).

2.3. Culture conditions

C. gallica KJ412304 was cultured in semi-solid-state fermenta-tion conditions in 250-mL cotton-plugged Erlenmeyer flasks con-taining 5.0 g of sawdust. The substrate was hydrated with 15 mL ofminimum medium (MM) adjusted by 25 mM acetate buffer (pH5.0). This MM contained (g L�1): glucose, 5; casein peptone, 6;KH2PO4, 0.025; MgSO4$7H2O, 0.25; KCl, 0.5. Inoculationwas carriedout directly in the Erlenmeyer flasks. Six plugs (diameter, 3 mm),from a 5-day growing fungus onmalt extract agar (MEA) plates, perErlenmeyer were used as inoculum. The cultures were supple-mented with CuSO4 as laccase-inducer (solution sterilized sepa-rately, 60 mM) at the beginning of the cultivation. The Erlenmeyerflasks were incubated statically for 12 days under an air atmosphereat 30 �C and 90% humidity, to avoid evaporation, in completedarkness.

At the end of cultivation (12 days) the flask contents wereextracted with sodium acetate buffer (pH 5.0, 25 mM) undershaking for 1 h, filtered and centrifuged at 7000 rpm for 20 min at4 �C. The supernatant was collected and used as enzyme source forquantification 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 QFFcolumn (GE Healthcare) pre-equilibrated with 25 mM sodium ac-etate pH 5.5 and the retained proteins were eluted with a 0e250 mM NaCl gradient (140 min, 1 mL min�1). The laccase activefractions were pooled, concentrated and dialyzed against the abovebuffer, 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 highimmobilization efficiency, the gelling agent (sodium alginate andcalcium chloride solution) and the quantity of enzyme introducedshould be studied and optimized. For this purpose varying con-centrations 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 immobilizationof 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)) wasprepared by dissolving sodium alginate in deionized water con-taining a certain amount of laccase (200e800 mg L�1). Thendifferent ratios of enzyme/alginate (E/A) (v/v) were mixed undershaking (Baysal, 2007). The mixture was dropped by means of asyringe into a CaCl2 solution (1e4% w/v) under shaking. After 1 hthe beads (about 3e4 mm in diameter) were collected from thesolution and washed with CaCl2 0.5% (w/v) twice and then washedthree times with deionized water. The filtered hardening solutionsand the two washings were collected for loading efficiency (Eq. (1))and immobilization yields determination (Eq. (2)). Loading effi-ciency and immobilized yield were defined as the percentage oftotal enzyme entrapped and the specific activity ratio of entrappedlaccase to free laccase, respectively.

Loading efficiency ð%Þ ¼h�

CiVi � CfVf

�.Cii� 100 (1)

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

Immobilization ð%Þ ¼ ððAi � AwashÞ=AiÞ � 100 (2)

Where, Ai is the initial activity of the free enzyme introduced intothe mixture of Ca-alginate solution assayed using ABTS [2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate andAwash is the laccase activity detected in the curing solution and thetwo washing solutions assayed using the same substrate.

All the immobilized laccase beads were kept in distilled water at4 �C until further use.

2.6. Enzyme assay and protein estimation

The activity of the free and immobilized laccase was assayedusing 1 mM ABTS as a substrate. For the free enzyme the reactionwas initiated by adding 0.3 mL of ABTS to a mixture consisting of3 mL of 100 mM succinic acid buffer (pH 5.0) and the enzyme(Erden et al., 2009). The molar extinction coefficient of ABTS is36000 M�1 cm�1. The increase in absorbance was recorded at

D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e78 73

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 ABTSwere incubated under shaking at 100 rpm for 5 min. Oxidation ofABTS was followed by an absorbance increase at 420 nm. One ac-tivity unit (U) of laccase was defined as the amount of enzymerequired to catalyze 1 mmoL mL�1 of substrate per minute. All theassays were carried out in triplicate.

Protein estimation of free laccase was performed using themethod of Bradford using the commercial reagent Bio-Rad (SigmaChemical, St. Louis, USA) and bovine serum albumin (BSA) as astandard (Bradford, 1976). For the immobilized laccase, the amountof protein in the supernatant solution after immobilization wasdetermined using the Bradford method. Bound proteins weredetermined as the difference between the initial and the residualprotein concentrations.

2.7. Characterization of the immobilized laccase

2.7.1. Stability testsThe effect of pH on laccase immobilized beads was compared

and studied by incubating the samples in 100 mM succinic acidbuffer ranging from pH 2.0 to 9.0 for 24 h. The residual activity wasestimated at the end of the incubation period for all the different pHranges as described in the Section 2.6.

Thermal stability was assayed by incubating the laccaseimmobilized beads and the free laccase simultaneously at 55 �C for210 min. The residual activity was measured as described in theSection 2.6.

Storage stability experiment was performed to determine thestabilities of free and immobilized laccase. For storage stabilitymeasurements, immobilized laccase was kept at 4 �C withoutbuffer. The activity of immobilized laccase was followed for 20 daysand determined by the laccase activity assay procedure reported inthe Section 2.6. Then, the immobilized laccase was reused each 2days and the variation of activity was measured with respect to theinitial one. After each assay, laccase immobilized beads werewashed with buffer and stored at 4 �C for further uses.

2.7.2. ReusabilityCa-alginate beads were used several times for the different dye

decolorization reactions to test the reusability of laccase entrapped

Table 1Characteristics of the synthetic dyes used.

Dye CI number CI name

Remazol Brilliant Blue R (RBBR) 61200 Reactive Blue 19

Reactive Black 5 (RB5) 20505 Reactive Black 5

Bismark Brown R (BBR) 21010 Basic Brown 4

Lanaset Grey G (LG) e e

CI: color index.

beads. Seven decolorization cycles of 24 h each were performed foreach of the different tested dyes. After each cycle, the beads wereremoved and washed with 50 mM Tris HCl buffer (pH 7.2) and thesolution replaced with fresh dye solution. The activity of freshlyprepared beads in the first runwas defined as 100%. The reusabilitystudy was performed in triplicate.

2.8. Decolorization studies of immobilized laccase on differentsynthetic textile dyes

Four synthetic dyes, Remazol Brilliant Blue R (RBBR), ReactiveBlack 5 (RB5), Bismark Brown R (BBR) and Lanaset Grey G (LG: com-plex metal dye) were selected as model dyes to study the decolor-ization ability of the C. gallica laccase immobilized into Ca-alginatebeads (Table 1). Stock solutions of the dyes (0.1% w/v in water) werestored in thedarkat roomtemperature. Experimentswereperformedusing 50-mL disposable flasks in a 5-mL final reaction volume. Thereactionmixture, containing 10% (v/v) beads/100mM succinic bufferpH5.0 and 1mMofHBT (in laccasemediator systems),was incubatedstatically in the dark at 30 �C. Dye concentrations were selected inorder to obtain around 1.5 absorbance units at the maximumwave-length of each dye in the visible spectrum (75 mg L�1 for RBBR,66.7 mg L�1 for RB5, 36.0 mg L�1 for BBR and 100.0 mg L�1 for LG). Acontrol reactionwithCa-alginate beadswithout laccasewaspreparedunder the same conditions to detect possible removal of color due todye adsorption onto the alginate beads. All the experiments wereperformed in duplicate. Dye concentrations were spectrophotomet-rically (Shimadzu UV 1650 PC) measured from 400 to 800 nm andcalculated by measuring the area under the plot. Decolorization wascalculated by the following equation:

Decolorization ð%Þ ¼�Absinitial � Absfinal

�.Absinitial � 100;

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

2.9. Data analysis

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

Class lmax Structure

Anthraquinonic 592

Diazo 597

Diazo 468

Complex metal dye 579 Not disclosed

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

Purificationsteps

Volume(mL)

Laccaseactivity(U)

Proteincontent(mg)

Specificactivity(U/mg)

Purificationfold

Yield (%)

Culture extract 250 1450 105 13.8 1 100Ultrafiltration 40 1080 31.5 34.2 2.4 74.4HiTrap QFF 200 980 16.4 59.7 4.3 67.5Ultrafiltration 40 880 12.8 68.7 4.9 60.6

D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e7874

Excel-software (Microsoft). Readings were considered significantwhen p was �0.05.

3. Results and discussion

3.1. Laccase production and purification

Laccase was produced from a locally-isolated strain of thewhite-rot-fungus C. gallica KJ412304 grown on sawdust asdescribed in 2.3. The cell-free extracellular liquid produced wassubjected to partial purification by ultrafiltration and columnchromatography as summarized in Table 2.

3.2. Laccase immobilization into alginate beads

The gelation of alginate could be initiated by mixing sodiumalginate and calcium chloride solution. However, the preparation ofbeads with rigidity and proper permeability for enzyme gelentrapment is based on both, the concentration of sodium alginate

Fig. 1. (a) Effects of alginate concentration. Immobilization conditions: Laccase solution (0.2concentration. Immobilization conditions: Laccase solution (0.5 mg/mL), alginate solution (Alginate ratio (v:v). Immobilization conditions: Laccase solution (0.5 mg mL�1), alginate (2

and the ability of calcium ions to cross link with sodium alginate,and also the ratio Enzyme/Alginate (E/A) (Phetsom et al., 2009). Thefollowing parameters depict the immobilization efficiency of thepurified 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) aredefined as described in Eqs. (1) and (2), respectively.

In Fig. 1a, the effect of various alginate concentrations (from 1 to4% (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 abovementioned figure, it is shown that the maximum entrapped laccaseoccurred at 0.5 and 1% (w/v) sodium alginate concentration owingto the larger pore size of the less tightly crossed linked fragile Ca-alginate beads. However, there was a gradual decrease in theimmobilization efficiency with 3% and 4% sodium alginate. Thismay be attributed to the pore size of the beads and the degree ofcross-linking of the gelling agent. Riaz et al. (2009) found that thelower concentration of sodium alginate solution leads to thegreater pore size of the beads resulting in increased leakage of theenzyme from the beads. Similarly, the higher the concentration ofsodium alginate, the smaller the pore size of the beads leading tolower immobilization efficiency. In addition, an increase in alginateincreases the viscosity of the solution making enzyme encapsula-tion cumbersome (Geethanjali and Subash, 2013).

From the Fig. 1a, it is observed that sodium alginate at a con-centration of 2.5% (w/v) registered the highest entrapped laccaseactivity with the loading efficiency value being 83 � 0.5%. This is inaccordance with the results of Geethanjali and Subash (2013), whostated that sodium alginate ranging from 2 to 3%, was suitable forimmobilization of protease.

e1 mg mL�1), CaCl2 solution (1% w/v), Enzyme/Alginate ratio (1:4); (b) Effects of CaCl22% w/v). Loading efficiency (bar) and immobilization yield (A); (c) Effect of Enzyme/% w/v), CaCl2 (2% w/v).

D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e78 75

Furthermore, calcium chloride is used as a cross linking agent,and its concentration affects the activity and the stability ofimmobilized enzyme. Fig. 1b shows that an increase in the calciumchloride concentration from 1.5 to 3% (w/v) at 2% (w/v) alginateconcentration had little effect on the loading efficiency (77.3� 1.1%,82.2� 2.4%, respectively). Former studies showed that the diffusionof high molecular weight substances from the Ca-alginate beadsinto the bulk solution was little affected by increases in the CaCl2concentration (1e2% w/v), but it was considerably limited by in-creases in alginate concentration (2e4% w/v) (Tanaka et al., 1984).Therefore, alginate concentration plays a key role for enzymeentrapment into Ca-alginate beads.

Moreover, the effect of ratio (E/A) on the loading efficiency andimmobilization yield is shown in Fig. 1c. Since the beads preparedin the present work were very small, their loading efficiency mightbe relatively limited which would cause a decrease in the immo-bilization yield at a higher E/A ratio (�1:8). The finding that theratio (E/A) of 1:4 was found to be the optimum for the immobili-zation of purified laccase from C. gallica can be contrasted with thereports of Lu et al. (2007), who reported that immobilization ofenzyme 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 effi-ciency and immobilized yield of the immobilized laccase were88.1 � 2.4% and 93.3 � 1.1%, respectively.

3.3. Characterization of the free and immobilized laccase

3.3.1. Stability testsThe thermal stability of the free and immobilized C. gallica lac-

case was investigated at 55 �C for different incubation times. Thistemperature was selected because it is the temperature normallyused in the textile industry for dyeing.

The data from the Fig. 2, show that the immobilized and freelaccase maintained 91.2 � 0.7% and 25.6 � 1.3% of their initialactivities, respectively, after 90 min of incubation at 55 �C. Theresidual activity at 210 min of incubation was about 67 � 0.8% forthe immobilized laccase and 3% for the free enzyme. This is inaccordance with the results of Reyes et al. (1999), who found thatan immobilized laccase from C. gallica UAMH8260 on activatedagarose showed higher thermal stability at 70 �C than the freeenzyme.

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

The pH stability curve of immobilized and free laccase isdepicted in Fig. 3. From the values in the above mentioned figure, itcan be deduced that compared to the free laccase, the immobilizedlaccase stability in acidic pH values was higher (about 30%). Also,the stability of the immobilized laccase in the pH range 7.0e9.0 wasabout 20% higher than the free enzyme.

Generally, free enzymes can lose their activities quickly (Ceviket al., 2011). Hence, it is advisable to immobilized enzymes. Stor-age stability is one of the most important parameters to beconsidered in biocatalyst immobilization.

Fig. 4 indicates that, at the end of the 20 days of storage, the freelaccase and immobilized laccase retained about 22.4 � 1.8% and82.7 � 0.9% of their initial activities, respectively. Similar findingswere observed by Huang et al. (2006), who reported that afterstorage at 4 �C for one month, the activity of Pycnoporus sanguineuslaccase immobilized on copper tetra-aminophthalocyanine-Fe3O4nanoparticle composite was 85% of its initial activity, while that ofthe free laccase was only 30%.

3.3.2. Decolorization of different synthetic dyes by free andimmobilized laccase

The dye-decolorizing potential of immobilized and free laccasefrom C. gallica was demonstrated for different textile dyesbelonging to 3 dye families: an anthraquinone dye (RBBR), diazodyes (RB-5 and BBR) and a complex metal dye (LG) (Fig. 5aed).

From data in Fig. 5a, it can be observed that RBBR was rapidlydecolorized by both the free and immobilized laccase alone,compared to LG, RB5 and BBR (Fig. 5bed). For the diazo and thecomplex metal dye, the maximum decolorization yields obtainedwere not higher than 50% within 24 h of incubation without redoxmediators, whereas theyweremore 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, thisanthraquinone dye was decolorized to 74.6% and increased up to90.3% using immobilized laccase after 90 min of treatment (Fig. 5a).Furthermore, the decolorization levels of RBBR did not improve byadding 1 mM of HBT (Fig. 5a). The above results are supported byMechichi et al. (2006), who emphasized no effect of the redoxmediator HBT at concentrations between 0.125 and 2.5 mM on thedecolorization of RBBR. Other results also reported that theoxidation 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 freelaccase is depicted in Fig. 5b. The results show a high percentage ofdye adsorption onto the beads (34.6 � 0.6%) within 24 h compared

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

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

D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e7876

to the other dyes studied (Fig. 5a, d). In the case of entrapped andfree enzyme, this azo dye was decolorized to 52.8 � 1.7% and47.1 �1.1%, respectively, within 24 h. Thus, the immobilized laccasewas not able to degrade the dye adsorbed onto the beads and dyedecolorization was mainly due to adsorption (Fig. 5b). The additionof HBT did no enhance significantly the color removal rates. Theseresults are in agreement with the study performed by Enayatzamiret al. (2010), who stated that the dye BBR was resistant tobiodegradation by the white-rot fungus P. chrysosporium immobi-lized into alginate beads and dye removal was mainly due to dyeadsorption onto the alginate beads. The higher affinity of BBR foradsorption onto alginate beads might be related to the differentionisation of this dye, since it is a cationic dye whereas the othertested dyes are anionic (Enayatzamir et al., 2010).

Fig. 5. Decolorization time-course of synthetic dyes (a) RBBR (b) BBR (c) LG (d) RB5 by imm

Fig. 5c illustrates the decolorization yields of the dye LG bylaccase and laccase-mediator system. LG decolorization performedwith immobilized laccase showed a removal percentage of49 � 0.4% in 24 h and the free enzyme of 30.5 � 3.4% for the sameperiod of time. In the presence of HBT as a redox mediatormaximum decolorization of 86.5 � 1.4% and 72.2 � 0.2% was ach-ieved after 24 h with the immobilized and the free enzyme,respectively. LG is a recalcitrant commercial mixture of metal-complex dyes that contains chromium (Crþ3) and cobalt (Coþ2)(Blánquez et al., 2004). Most of the previous studies on LG decol-orization involved whole fungal cultures (Blánquez et al., 2004,2007; Gabarrell et al., 2012; Daâssi et al., 2013) but there are nostudies with laccase or laccase-mediator system. In the presence ofHBT (1 mM) the immobilized enzyme exhibited efficient LGdecolorization (86.5%) after 24 h.

In the case of the azo dye RB5, the immobilized enzyme showedaround 31% decolorization in 8 h and 24 h of incubation withoutHBT. However, the addition of 1 mM HBT enhanced the decolor-ization nearly to 1.3-fold by the immobilized enzyme, and, thus, amaximum decolorization of 78.2 � 0.7% was observed within 8 h(Fig. 5d). Kunamneni et al. (2008), found a similar result in thedecolorization of RB5 by a laccase fromMyceliophthora thermophila.

Generally, the mechanism of dye removal using immobilizedenzyme or cells may be due to either enzymatic biodegradation orbioaccumulation/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 dyeadsorption onto the alginate beads, a control reaction with Ca-alginate beads without laccase was prepared. Dye removal mech-anism can also be judged clearly by inspecting the color of thebeads up to the treatment dye process. It was observed that thealginate beads became colored especially after contacting with BBRand LG. From the Fig. 5, it is clearly understood that the BBR and LGremoval mechanism is essentially biosorption of the dye onto

obilized or free laccase in the presence and absence of redox mediator (HBT, 1 mM).

D. Daâssi et al. / International Biodeterioration & Biodegradation 90 (2014) 71e78 77

alginate beads, with percentages of 34% and 24%, respectively,compared to the other dyes studied. Ca-alginate beads were able toremove only 12% and 17% of RBBR and RB5 dye, respectively. Thus,the predominant mechanism involved in RBBR and RB5 removalwas laccase biodegradation.

3.3.3. ReusabilityIn this stage of research, it was investigated whether the

alginate-immobilized C. gallica laccase could be successfully re-used after storage at 4 �C. Reusability of immobilized enzymes inthe biodegradation process exhibits the most important aspect forindustrial applications, since it decreases the cost of the process.Thus, the reusability of the immobilized laccase in seven successivebatches of 24 h each was investigated. The relative decolorizationrates are depicted in Fig. 6. From data in the above figure, it can bedetected that after the 4th cycle, the relative decolorization valuesfor 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 cycleshas been explained differently in the literature. This may be relatedwith enzyme inactivation. Indeed, upon repeated uses, the blockingof some pores of beads by substrate or product may take place. Thisrestriction may cause a decrease in the efficient activity of C. gallicalaccase entrapped into the gel after successive decolorization cycles.However, Anwar et al. (2009) emphasized that the decrease in ac-tivity occurred on further reusemay be due to the leakage of enzymefrom alginate beads during washing at the end of each cycle.

In the literature, there are several articles reporting the suc-cessful reuse of various immobilized laccase systems. Thus, a lac-case from Panus conchatus immobilized on activated polyvinylalcohol retained 60% of its activity after ten batch uses and morethan 50% after 17 batch uses (Yinghui et al., 2002) and a laccasefrom P. sanguineus immobilized on copper tetraaminoph-thalocyanine (CuTAPc)-Fe3O4 magnetic nano-composite retained80% of its initial activity after 5 bath uses (Xiao et al., 2006).

4. Conclusions

From the current study it can be concluded that the immobili-zation of laccase into Ca-alginate beads improved its thermal andstorage stabilities. Thus, the immobilized and free laccase main-tained 91.2% and 25.6% of their initial activities, respectively, after90 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 decolorizationof: LG (>) BBR (,) RBBR (6) RB5 (B). Data were mean values � SD. 7 decolorizationcycles, 24 h each.

the immobilized and free laccase retained about 82.7% and 22.4% oftheir initial activities, respectively. In addition, the immobilizedlaccase exhibited efficient textile dye decolorization in severalsuccessive batches. This would provide economical advantageswhen used in large-scale applications.

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