Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 7003–7010

Fungal laccase, cellobiose dehydrogenase, and chemicalmediators: Combined actions for the decolorization

of different classes of textile dyes

Ilaria Ciullini, Silvia Tilli, Andrea Scozzafava, Fabrizio Briganti *

Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Universita degli Studi di Firenze, Via Della Lastruccia 3, 50019 Firenze, Italy

Received 5 November 2007; received in revised form 4 January 2008; accepted 9 January 2008Available online 20 February 2008

Abstract

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among themost utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude cul-ture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger differentlevels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorizesome dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and thechemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes andtheir electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to theactivity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyesproving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Trametes trogii; Laccase; Cellobiose dehydrogenase; Textile dyes; Decolorization

1. Introduction

White rot fungi, a heterogeneous group of organisms arecapable of degrading lignin and the other main wood com-ponents, fundamental for carbon flux in ecosystems. Theirbiodegradation capacities are due to highly non-specific,free-radical-mediated processes resulting from the activitiesof several enzymes secreted by these fungi such as laccase,manganese peroxidase (MnP) and lignin peroxidase (LiP)(Fu and Viraraghavan, 2001). These enzymatic systemsenable white rot fungi to degrade a wide range of pollu-tants, including polycyclic aromatic hydrocarbons (PAH),polychlorinated biphenyls (PCB), pesticides, explosives,synthetic polymers and synthetic dyes (Pointing, 2001).

0960-8524/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2008.01.019

* Corresponding author. Tel.: +39 0554573343; fax: +39 0554573333.E-mail address: fbriganti@unifi.it (F. Briganti).

Synthetic dyes are being increasingly used in the textile,paper, pharmaceutical, cosmetics and food industries. Over7 � 105 tonnes of approximately 10,000 different dyes andpigments are produced annually worldwide, of whichabout 50,000 tonnes are discharged into the environment(Lewis, 1999). The discharge of very small amounts of dyes(less than 1 ppm for some dyes) is aesthetically displeasing,impedes light penetration, affects gas solubility damagingthe quality of the receiving streams and may be toxic totreatment processes, to food chain organisms and to aqua-tic life. For these reasons several countries are adoptingstringent regulations for the release of colored industrialeffluents. Azo, anthraquinone and indigo are the majorchromophores found in commercial dyes. Decolorizationof these dyes by physical or chemical methods is financiallyand often also methodologically demanding, time-consum-ing and mostly not very effective. Because of the range ofchemical structures and properties, the degradation of mol-

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ecules of dyes in the environment by microorganisms isvery slow (Pierce, 1994). Moreover, the industrially impor-tant azo dyes are cleaved under anaerobic conditions bybacterial azo-reductases to the corresponding amines,many of which are mutagenic and/or carcinogenic generat-ing potential health hazards (Banat et al., 1996).

At present, a number of studies have focused on the uti-lization of fungi since their mechanisms of dyes decoloriza-tion involve oxidative reactions which therefore do notproduce toxic amines. However, using fungal biomass orsingle enzymes to remove color in a dye wastewater is stillin the research stage. Recent studies have also shown thatcellobiose dehydrogenase (CDH hereafter), an extracellularhaemo-flavo-enzyme, produced by a number of wood-degrading and phytopathogenic fungi, has a role in theearly events of lignocelluloses degradation and wood colo-nization (Henriksson et al., 2000), due to its ability to facil-itate the formation of free hydroxyl radicals. CDH hasbeen reported to display in vitro a synergism with laccasesin the decolorization of an anthraquinonic dye, anddirectly in the oxidation of several chemicals (Vanhulleet al., 2007).

Funalia (Trametes) trogii is a widely distributed whiterot basidiomycete, good producer of laccases and other lig-ninolytic enzymes (Levin et al., 2005). A few studies on itscapabilities in the decolorization of only some dyes havealso been reported (Colao et al., 2006; Levin et al., 2001).The F. trogii strain 201, subject of this research, secreteslaccases and no peroxidases under the different growth con-ditions utilized in this study and we here report for the firsttime that in the presence of cellulose besides laccases alsoCDH is secreted in abundance.

In this paper, we report the investigation of the activitiesof the redox enzymes secreted by F. trogii 201 under differ-ent growth conditions on the decolorization of several clas-ses of textile dyes among the most diffused in textileapplications. The combined utilization of CDH or redoxmediators with laccase was also investigated for the decol-orization of the most recalcitrant dyes.

2. Methods

2.1. Chemicals

All the chemicals were purchased from Sigma ChemicalCo. Agar and Yeast Extract were from Oxoid Ltd. Textiledyes utilized were from Eurocolor S.p.A.; InternationalColor S.p.A.; Ciba Specialty Chemicals S.p.A.; Kem.Color S.p.A; Novacolor s.r.l; or from AlphaColor S.p.A.

2.2. Organism and culturing conditions for laccase

production

The white rot fungus F. trogii 201 (DSM 11919) wasmaintained on basidiomycete rich medium (BRM) (Bezalelet al., 1997) agar plates at 4 �C and periodically transferredonto fresh BRM agar plates and grown at 28 �C. After 4–6

days of growth on agar plates, 500 ml shaken flask culturescontaining 150 ml liquid BRM were prepared and inocu-lated with 10 plugs of fungal mycelia (about 25 mm2) andgrown in the dark at 28 �C under continuous stirring at130 rpm. After 4 days the grown mycelia were transferredin baffled 2000-ml Erlenmeyer flasks, closed with sterileair permeable silicon corks, containing 1000 ml of freshBRM liquid medium and grown under the same condi-tions. The laccase expression was induced by the additionof 150 lM CuSO4 to the starting medium. Other inducers(veratryl alcohol 0.25 mM or Cu(II) 0.15, 0.5 or 1.0 mM)were added after 2 days of incubation. When the extracel-lular laccase activity reached a maximum about on day 7,the culture supernatant was collected by filtration throughWhatman No. 1 paper and concentrated using an ultrafil-tration Vivaflow 200 module (Sartorius group) with a30,000 Da cut-off membrane.

2.3. Culturing conditions for simultaneous cellobiose

dehydrogenase (CDH) and laccase production

The fungus F. trogii 201 was cultivated in the abovedescribed conditions but on modified BRM where10 g l�1 of microcrystalline cellulose powder was added ascarbon source instead of glucose to obtain the simulta-neous production of CDH and laccase. When the extracel-lular CDH activity reached a maximum, about on days 8–9, the culture supernatant was collected by filtration andconcentrated as reported above.

2.4. Enzyme assays

Laccase activity was determined spectrophotometricallybased on the capacity of this enzyme to oxidize the non-phenolic compound 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (e420 = 36,000 M�1 cm�1), pH 3 at25 �C; 1 U of laccase activity was defined as the amountof enzyme oxidizing 1 lmol substrate/min.

CDH activity was assayed by following the decrease inabsorbance of the electron acceptor, i.e. 2,6-dichlorophe-nol-indophenol (DCIP), at 520 nm (e520 = 6.8 � 103

M�1 cm�1), pH 4.0 and 37 �C. One unit of enzyme activityis defined as the amount of enzyme reducing 1 lmol DCIP/min under the above reaction conditions. The combineddetermination of laccase and CDH activities was per-formed using 0.1 mM DCPIP following the method byVasil’chenko et al. (2005). To understand if the DCPIPreducing activity observed was really due to CDH andnot to a sugar oxidase usually present in the cellulolytic sys-tems of fungi, i.e. glucose oxidase, in this assay we substi-tuted cellobiose with D-glucose; no glucose oxidaseactivity was observed.

MnP activity was estimated by the formation of Mn3+–tartrate complex (e238: 6500 M�1 cm�1) at pH 5, 25 �C. LiPactivity was determined by the H2O2-dependent veratralde-hyde (3,4-dimethoxybenzaldehyde) formation (e310 = 9300M�1 cm�1), pH 3, 25 �C.

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2.5. Laccase purification

The crude protein pellet was obtained by solid(NH4)2SO4 addition up to 80% (w/v) to the extracellularfungal extract; then it was dialyzed against 10 mM sodiumphosphate buffer (pH 6.0) and concentrated by ultrafiltra-tion (membrane cut-off 30,000). All the following chro-matographic steps were performed utilizing a HPLCWaters system composed by a 600 Solvent Module and a996 diode array UV–vis detector interfaced to a personalcomputer running a Millennium chromatographic system.The laccase was then purified through the followingsequential chromatographic steps: (1) DEAE macro prepion exchange column (Bio-Rad, 2.6 � 14 cm), (2) Phenyl-Sepharose HP column (Pharmacia Biotech, 1.6 � 10 cm),(3) Q10 ion exchange column (Bio-Rad, 1.2 � 8.8 cm),and (4) Superdex 75 column (Pharmacia Biotech,1.6 � 62,5 cm). The fractions containing laccase with aA280/A600 ratio lower than 13, index of a purity larger than99%, were pooled, concentrated and further used.

2.6. Enzymatic dyes decolorization

The reaction mixtures for dyes-decolorizing activity wereprepared in 50 ml shaken flasks and consisted of an aqueoussolution of dye, 0.5 mg ml�1 (0.5–1.1 mM) with the excep-tions of Acid Blue 324: 0.3 mg ml�1 (0.7 mM), Acid Red374: 0.04 mg ml�1(0.05 mM) and Acid Yellow 129:0.08 mg ml�1 (0.25 mM) (due to their lower water solubili-ties) in a total volume of 20 ml. The reactions were initiatedadding crude extract or pure laccase and incubated at 30 �Cwith shaking (300 rpm) for the appropriate times. Samplesof dye solutions were taken at regular times, centrifugedat 13,000 rpm for 5 min to eventually remove suspendedparticles and decolorization was measured after appropri-ate dilution. The dyes partially or non-decolorized by lac-case were tested with the combined action of laccase and4 mM 1-hydroxybenzotriazole (HBT) used as a redox medi-ator, the dye solution was adjusted to pH 5 using 0.1 Msodium citrate buffer. These dyes were also tested for decol-orization with the combined action of laccase and CDHactivities at pH 7. UV measurements were carried out ona double beam Perkin–Elmer EZ 301 spectrophotometerusing 1 cm path length Hellma 110 quartz suprasil cellsthermostated with a Lauda thermostat RE112. The UV/vis absorption spectrum was recorded (300–800 nm) foreach dye and decolorization was followed monitoring theabsorption at the maximal peak wavelength.

3. Results

3.1. Optimization of enzymatic activities of laccase and

CDH

F. trogii in vitro laccase production reached a maximumon days 7–9 of cultivation. It was stimulated by the addi-tion of CuSO4 or veratryl alcohol to the growth medium.

Activity increases from 2.0 U ml�1 to 4.0, 6.0 or 7.5 U ml�1

were observed when the copper concentration was raised,after 2 days of growth, by the further addition of 0.15,0.50, and 1.0 mM CuSO4, respectively. The addition of0.25 mM veratryl alcohol (instead of Cu(II)) after 2 daysof growth resulted in an increase of laccase production to7.2 U ml�1, comparable to 7.5 U ml�1 obtained with thesupplement of 1.0 mM CuSO4. Detectable CDH produc-tion occurred only in media containing cellulose powderas carbon source. For such purpose the BRM mediumwas modified by substituting glucose, which inhibitsCDH expression, with 10 g l�1 cellulose (Stapleton andDobson, 2003). The best CDH production was obtainedon days 8–9 of cultivation (up to 0.6 U ml�1). At the sametime the laccase activity reached about 5–6 U ml�1. MnPand LiP activities were not detected in any of the differentconditions utilized for fungal growth.

3.2. Dyes decolorization with crude extracellular extract and

pure laccase

A series of textile dyes was selected on the basis of theirextensive utilization in dyeing factories. Several mono-azo(Acid Yellow 49, Acid Red 42, Reactive Yellow 39, ReactiveRed 272), chromo-complexed mono-azo (Acid Blue 158,Acid Black 194, Acid Yellow 129, Acid Red 186), disazo(Acid Black 1, Direct Red, 243, Acid Red 374), tri-azo(Direct Blue 71), poly-azo (Direct Black 22) and anthraqui-nonic dyes (Reactive Blue 69, Acid Blue 80, Acid Blue 324)were tested for decolorization utilizing a crude extracellularculture extract of F. trogii containing laccase activity(1.5 U ml�1). The time courses for the dyes decolorizationswere followed for at least 3 days but the maximal effectwas substantially reached within 24 h for all the dyes tested.The initial experiments were performed at pH 3.0 since themain laccase from F. trogii showed the maximal activity atsuch pH value (Garzillo et al., 2001). As shown in Table 1,at such pH only the dyes which had adequate water solubil-ity were investigated. The chromo-complexed azo Acid Blue158, Acid Black 194, Acid Red 186, the disazo Acid Black 1,and the anthraquinonic Reactive Blue 69 were decolorized toa large extent (>85%) (see Table 1), whereas for the mono-azo Reactive Yellow 39, Reactive Red 272, and the disazoDirect Red 243 only 17–28% decolorization was achieved.

All the dyes were subsequently tested at pH 7.0 wherethey were all soluble. In particular, the dyes insoluble atpH 3 gave the following results: the mono-azo Acid Red42, the disazo Acid Red 374, the anthraquinonic Acid Blue324 and Acid Blue 80 were largely decolorized (see Table 1)but the mono-azo Acid Yellow 49, the mono-azo chromocomplexes Acid Yellow 129, the tri-azo Direct Blue 71,and the poly-azo Direct Black 22 resulted in a lower decol-orization yield. Regarding the dyes tested at both pH 3 and7, we noted comparable or even better decolorizationresults at pH 7.0 using the same amount of laccase,although the main laccase from F. trogii shows about 10times reduced activity towards ABTS or DMP at that pH

Table 1Decolorization (%) of different classes of textile dyes by Funalia trogii laccase

Class Dyes pH 3 pH 7 HBT, pH 5.0 CDH, pH 7.0

Monoazo Acid Yellow 49 – 23.3 35.4 96.3 (26.3)a

Acid Red 42 – 93.4 n.d. n.d.Reactive Yellow 39 21.0 0.0 81.6 87 (74.2)a, pH 3

0.0, pH 7Reactive Red 272 21.4 15.9 79.5 69 (27)a

Monoazo chromo complexes Acid Blue 158 82.4 99.3 n.d. n.d.Acid Black 194 99.0 88.7 n.d. n.d.Acid Yellow 129 – 27.5 23.5 70 (35)a

Acid Red 186 96.6 96.5 n.d. n.d.

Disazo Acid Black 1 85.0 95.0 n.d. n.d.Direct Red 243 28.2 90.4 92.0 n.d.Acid Red 374 – 100.0 n.d. n.d.

Tri-azo Direct Blue 71 – 21.5 91.5 97.4 (32.5)a

Poly-azo Direct Black 22 – 31.3 64.3 100.0 (42)a

Anthraquinonic Reactive Blue 69 96.7 91.1 n.d. 100.0 (100)a

Acid Blue 80 – 98.6 n.d. n.d.Acid Blue 324 – 75.0 85.0 95 (75)a

a The data in parenthesis are the results of the decolorization experiments performed utilizing the fungal extract containing CDH plus laccase activitieswithout the addition of lactose. No quantification of the oligosaccharides content was performed on such extracts.

0 10 20 30 40 50

Abs

orba

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0.2

0.4

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pH 7.0

Time (hours)0 10 20 30 40 50 60

Abs

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0.0

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A

B

Fig. 1. (A) Effect of pH on the decolorization of Direct Red 243 utilizingcrude extracellular culture extract of F. trogii: containing laccase activity;pH 7.0 (s), and pH 3.0 (d) at 30 �C. Mean and standard deviation valuesof three replicates are shown. (B) Time course of the decolorization of

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(Garzillo et al., 2001). The largest difference was observedfor the disazo Direct Red 243 which showed a three timesincrease of decolorization at pH 7.0 (pH 3.0: 28.2%, pH7.0: 90.4%) (Fig. 1A).

The effect of decreasing the amount of starting laccaseactivity was tested on a chromo-complexed mono-azodye, Acid Black 194: reducing the laccase amount from1.5 to 1.0 U ml�1 did not substantially change the extent(99%) and the course of decolorization; further reductionsto 0.75 and 0.375 U ml�1 resulted in decreased levels ofdecolorization (91% and 84%, respectively, at 24 h).

Decolorizations by purified laccase and crude extracellu-lar fungal extract were also compared. Acid Black 194 wascompletely decolorized by the crude extract, whereas theapplication of the same amount of pure laccase resultedin a 77% decolorization only. The anthraquinonic ReactiveBlue 69 was instead decolorized by the same extent withboth pure enzyme and crude extract (�95%).

All the dyes totally or partially decolorized by the cata-lytic action of extract or pure laccase in the course of thepresent study did produce extensive precipitation of poly-merized products, within 24 h, easily eliminated by decant-ing, filtering or through low speed centrifugation.Furthermore, as previously suggested, laccase oxidationmight detoxify azo dyes because this reaction releases azolinkages as molecular nitrogen, impeding toxic aromaticamine formation (Chivukula and Renganathan, 1995).

Reactive Red 272 obtained with the addition of crude extracellular cultureextract of F. trogii: containing laccase (d) and laccase plus CDH and30 mM lactose (.) activities, pH 7.0, 30 �C. Control (s). Mean andstandard deviation values of three replicates are shown.

3.3. Dyes decolorization with the addition of HBT mediator

All the selected dyes which were not decolorized to alarge degree utilizing the sole crude fungal extract werethen subjected to tests employing the synthetic mediatorHBT, one of the best radical mediators for the laccase sys-

tem (Reyes et al., 1999). The laccase/HBT system is gener-ally more effective than laccase alone, since the free-radicalHBT species formed by the action of laccase on reduced

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HBT is a stronger oxidant than laccase itself (redox poten-tial of F. trogii laccase 760 mV at pH 7.0, and of radicalHBT: 1084 mV) (Garzillo et al., 2001; Zille et al., 2004).The mono-azo dyes Reactive Yellow 39 and ReactiveRed 272, the tri-azo Direct Blue 71, the poly-azo DirectBlack 22, recalcitrant to the sole action of laccase, andthe anthraquinonic dye Acid Blue 324 subjected to theaction of the laccase/HBT system, resulted in an extensivedecolorization within 4 h. On the contrary the mono-azoAcid Yellow 49 and the mono-azo chromo-complex AcidYellow 129 were substantially decolorized to the sameextent than with laccase activity alone (see Table 1).

3.4. Dyes decolorization with the addition of CDH

As a further alternative to the utilization of expensivechemical mediators the action of laccase was combined tothat of CDH, an enzyme able to indirectly generate in a Fen-ton type reaction hydroxyl radicals, very potent oxidants.CDH was expressed by F. trogii only when grown on mediacontaining cellulose and no glucose, as reported in Section 2.In this medium the fungus secreted CDH but also laccaseactivities. The decolorizing action of crude extracellular cul-ture extracts containing CDH (0.45 U ml�1) and laccase(1.5 U ml�1) activities was compared to that of extracts con-taining laccase activity only (1.5 U ml�1). The experimentswere performed at pH 7.0 with the exception of ReactiveYellow 39 which was tested both at pH 7.0 and 3.0. Further-more, since the action of CDH is associated to the oxidationof cellobiose, lactose, or similar carbohydrates, decoloriza-tion experiments with extracts containing CDH were per-formed in the absence or in the presence of 30 mMlactose. This carbohydrate was essential to activate theCDH to the production of hydrogen peroxide which furthergenerates, in a Fenton type reaction, hydroxyl radicals (seeSection 4). The presence of CDH plus lactose results in animproved reduction of color intensity for all the dyes tested,much higher than with laccase activity alone (Table 1). InFig. 1B is shown the time course of the decolorization ofReactive Red 272 obtained with the addition of crude extra-cellular culture extract of F. trogii containing laccase aloneand laccase plus CDH and 30 mM lactose. The additionof CDH yields a 4.3 times improvement in Reactive Red272 decolorization. Also the anthraquinonic dyes ReactiveBlue 69 and Acid Blue 324 were decolorized to a higherextent when CDH was utilized. Experiments performedadding lower concentrations of lactose (3 mM) or theCDH extract without lactose resulted in improvements indyes decolorization but to lower extents with respect toCDH plus 30 mM lactose (see Table 1).

4. Discussion

4.1. Enzymes induction

White rot basidiomycetes, involved in wood decayworldwide, and among them several different strains of F.

trogii have been shown to express several ligninolyticenzymes mainly laccases as well as lignin and manganeseperoxidases (Levin et al., 2002). Under the conditionsdescribed here, F. trogii 201 produces a major phenol oxi-dase (Colao et al., 2006; Garzillo et al., 1998, 2001); it doesnot produce any observable peroxidase activity. Inductionof laccase activity utilizing copper ions or lignin derivedaromatic compounds such as veratryl alcohol allowedmore than a three-fold increase in laccase activity (Levinet al., 2002; Palmieri et al., 2003).

CDH, another enzyme thought to be involved in ligno-cellulose degradation, is detected in F. trogii 201 when glu-cose is absent from the cultural medium and substituted bycellulose as previously observed for some white rot, brownrot and soft rot fungi (Stapleton and Dobson, 2003).

4.2. Dyes structure and biodegradability using laccase

The decolorization of various dyes with different struc-tural patterns was investigated using crude extracellularculture extract and pure laccase from F. trogii. Our systemwas able to efficiently degrade a number of commercial tex-tile dyes at pH 3.0. The decolorization of the antraquinonicdyes by laccase was expected, since high potential laccaseshave been shown to decolorize anthraquinonic dyes moreefficiently than other classes of dyes (Champagne andRamsay, 2005). Nevertheless, the most employed dyesbelong to the azo class which accounts for the 70% of alltextile dyes produced. It has been observed that laccasesdisplay substrate specificities and the chemical structuresof the dyes mainly due to differences in electron distribu-tion, charge density as well as steric hindrances largelyinfluence their decolorization extent and rates (Chivukulaand Renganathan, 1995; Pasti-Grigsby et al., 1992). Lac-cases modify azo dye structures by destroying their chro-mophoric assemblies, phenoxyl radicals are generated inthe reaction course (Chivukula and Renganathan, 1995).In a first step, one electron is abstracted from the pheno-lic/naphtholic ring, yielding a phenoxy radical; the abstrac-tion of a second electron, generates an aromatic cationwhich can be stabilized by electron-donating groups pres-ent in the ring. Actually the best biochemical decoloriza-tions were previously achieved with those azo dyes thatcarried hydroxyl groups, strong electron donating moieties,in ortho and para positions to the azo bond (Kandelbaueret al., 2004). The meta-substituted analogues were notattacked by laccase alone since the activating strength islower in such position. Electron withdrawing substituentssuch as halogen or nitro groups on the aromatic rings,make it difficult for oxidases to form cation radicals thusinhibiting dyes degradation. Instead, azo dyes character-ized by even weakly electron-donating methyl groups weredecolorized efficiently (Pasti-Grigsby et al., 1992). Further-more, heterocyclic azo dyes, containing pyrazole or triazolerings, were not decolorized significantly unless were presenthydroxyl and other electron donating groups on the hetero-cyclic and vicinal aromatic rings, in ortho position to the

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azo bond. Other effects, such as those caused by reactionintermediates, may contribute as well (Kandelbauer et al.,2004).

In the present study, all the mono-azo chromo-com-plexed dyes tested at pH 3.0 were extensively decolorized(Acid Blue 158, Acid Black 194, and Acid Red 186) sincethe oxidation was promoted by the hydroxyl groups inortho positions to the azo bonds on the vicinal rings.Instead, the mono-azo dyes checked at pH 3.0 (ReactiveYellow 39 and Reactive Red 272) resulted to be recalcitrantto decolorization because heterocyclic rings with chlorineatoms on them or in the surrounding aromatic rings as wellas sulfonic groups in ortho position to the azo were present.Regarding the two disazo dyes examined at pH 3.0 AcidBlack 1 was 85% decolorized, whereas Direct Red 243was degraded to a lower extent (Table 1) for the presenceof a central heterocyclic pyrazole ring and a higher numberof sulfonic groups in ortho to the azo bond in Direct Red243.

The crude extracellular culture extract of F. trogii uti-lized at pH 7.0 yielded high decolorization of all theanthraquinonic dyes tested (Reactive Blue 69, Acid Blue324 and Acid Blue 80). Among the mono-azo dyes testedonly Acid Red 42 was largely decolorized, whereas AcidYellow 49, Reactive Yellow 39 and Reactive Red 272 weremostly recalcitrant to degradation due to the presence ofelectron-withdrawing halogen atoms and sulfonic groupsin ortho positions to the azo bonds and heterocyclic rings,whereas in the Acid Red 42 a hydroxide and an aminogroup activate the azo bond attack. Regarding the monoazo chromo complexes the Acid Blue 158, Acid Black194 and Acid Red 186 were all degraded to large extentsdue to two activating hydroxyl groups in ortho to the azobonds either in homo- or hetero-cyclic rings; absent inthe poorly decolorized Acid Yellow 129 (Table 1) whichalso carries a deactivating carboxyl group. All the disazodyes tested in our experiments were decolorized at pH 7.0since they showed at least one hydroxyl group in orthoposition with respect to the azo bond. The Acid Black 1and particularly the Direct Red 243 were better decolorizedat pH 7.0, than at pH 3.0 (Fig. 1A). On the contrary the tri-azo Direct Blue 71, and the poly-azo Direct Black 22 werescarcely decolorized probably due to the higher number ofazo bonds surrounded by scarcely activated aromatic ringsand also to steric factors due to the large hindrances ofthese two molecules (Table 1).

4.3. Redox potential and biodegradability using laccase

All the structural differences mentioned above result insubstantial electron distribution and charge density varia-tions influencing the redox potential of the dyes. A correla-tion between the enzyme redox potential and its activitytoward substrates has been described (Xu et al., 1998)and the driving force for the redox reaction catalyzed bylaccases is expected to be proportional to the differencebetween the redox potentials of the oxidizing enzyme and

the reducing substrate (dye). A lower Eo of substrateand/or a higher Eo of laccase normally results in a higherrate of substrate oxidation and a linear correlation betweenthe percentage decolorization of each dye and the respec-tive redox potential was found (Zille et al., 2004).

The redox potential of phenolic or naphtholic substratesdecreases when pH increases since the mechanism of oxida-tive proton release is favored at high pH. According to Xu,a phenolic compound shows a decrease of Eo equal to0.059 V for each pH unit increased (at 25 �C), therefore apH change from 3 to 7 would result in an Eo (phenol)decrease of about 0.24 V (Xu, 1997). Over the same pHrange, the Eo change for laccases is generally much smaller(about 0.03 V for the main laccase from F. trogii) (Garzilloet al., 2001). Such pH dependences would then result in alarger difference in redox potential between phenolic sub-strates and laccase at higher pH, leading to an activityincrease for phenols oxidation as the pH increases (Xu,1997). This explains why the dyes tested at both pH 3and 7 are usually decolorized at better levels at pH 7.0:the disazo Direct Red 243 presents a three times increaseof decolorization at pH 7.0 with respect to pH 3.0(Fig. 1A). Also the disazo dye Acid Black 1 and the monoazo chromo-complexed Acid Blue 158 were better decolor-ized at pH 7.0. All of them exhibit phenolic/naphtholicrings directly connected to the azo moieties. Contrarily towhat generally observed in the literature our laccase systemhas been able to decolorize not only the anthraquinonicdyes but also a substantial part of the azo dyes tested.

4.4. Further observations

It has been previously noticed that over long periods ofoxidation, there can be a coupling between the reactionproducts, and even polymerization. It is known that lac-cases can catalyze the polymerization of various halogen-,alkyl-, and alkoxy-substituted anilines as well as phenolic,naphtholic, and aminophenolic compounds (Aktas andTanyolac, 2003). Contrarily to what observed in the inves-tigation by Zille et al. where soluble polymerized productsprovide unacceptable color levels in effluents (Zille et al.,2005), in all the decolorization performed in the presentstudy, polymerization generated substantial precipitationof the dyes within 24 h.

A comparison between the action of purified laccase andof crude extracellular extract showed that, whereas thecrude extract completely decolorized Acid Black 194; purelaccase application resulted in only 77% decolorization;possibly the presence of natural mediators in the extracel-lular fungal culture broth favours dye decolorization.

4.5. Decolorizations utilizing the laccase/HBT combination

Those dyes that were scarcely decolorized by laccasealone were subjected to the combined action of laccaseand HBT mediator which was previously shown to becapable to extend the oxidation power of laccases. Laccases

I. Ciullini et al. / Bioresource Technology 99 (2008) 7003–7010 7009

oxidize redox mediators forming short-lived cation radi-cals, which co-oxidize the substrate. These cation radicalscan be formed by two mechanisms: the redox mediatorcan perform either a one-electron oxidation of the sub-strate (Bourbonnais et al., 1998) or it extracts an H-atomfrom the substrate (Fabbrini et al., 2002). HBT operatesaccording to the second mechanism and the free-radicalproduced is an oxidant stronger than laccase itself(+1.084 V) (Reyes et al., 1999). Thus, the laccase/mediatorsystem is able to decolorize dyes having higher redoxpotentials than laccase alone (Claus et al., 2002). Amongthe dyes tested in the present study all the molecules recal-citrant to decolorization by laccase alone were degradedwith HBT, only exceptions Acid Yellow 49 and Acid Yel-low 129 (see Table 1). Probably the presence on both dyesof heterocyclic (pyrazole) rings flanking the azo bond onone side and on the other side aromatic rings containingonly electron withdrawing substituents make these dyesresistant even to the mediator attack.

4.6. Decolorizations utilizing the laccase/CDH combination

CDH is an extracellular fungal flavocytochrome enzymesecreted by several wood degrading fungi. While its physi-ological function is not clearly known, it is capable to pref-erentially oxidize oligosaccarides like cellobiose,cellotriose, or lactose to the corresponding lactones usinga wide spectrum of electron acceptors; among them thereduction of Fe(III) to Fe(II) and O2 to H2O2 can produce,by a Fenton type reaction, highly reactive hydroxyl radicals(Cameron and Aust, 2001; Henriksson et al., 2000). Theattack of hydroxyl radicals generated by CDH can yieldthe demethoxylation and/or hydroxylation of many aro-matic compounds, possibly leading to the conversion ofnonphenolic structures to phenolic ones, thus renderingthe molecule easily oxidized by laccases or peroxidases(Hilden et al., 2000).

In the present investigation, we observed enhancementsin the decolorization of dyes recalcitrant to laccase due tothe further addition of CDH and lactose; the latter neededas the CDH reducing substrate. When we utilized the crudeextract from culture broths modified for the production ofCDH and thus initially containing cellulose we alsoobtained decolorization enhancements without the addi-tion of lactose. This could be due to the presence of cellu-lose derived cellobiose or other CDH reducing substrates inthe extract.

Even in the case of hydroxyl radicals produced by CDHthe reaction rate has been reported to depend on the basicstructure of the molecule and on the nature of auxiliarygroups attached to the aromatic nuclei of the dyes (Galin-do and Kalt, 1999). In the present study, the addition ofCDH resulted in substantial increases in decolorizationfor all the dyes recalcitrant to the action of laccasealone. The combined action of laccase and CDH activatedby lactose always yielded decolorization values above69%.

We can therefore conclude that raw fungal extractscould be utilized in place of more expensive chemical treat-ments and synthetic mediators; in particular if the action oflaccase is supported by the formation of hydroxyl radicalstriggered by enzymes such as cellobiose dehydrogenase.

Acknowledgements

We thank the Assessorato all’Istruzione, Formazione eLavoro, Regione Toscana and we gratefully acknowledgethe support of Progetto MECHOS, POR Ob. 3 2000/2006 Toscana, Progetti integrati di ricerca Mis. D4 (Decre-to Regionale 03/04/2007 no. 1785).

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