Fungal laccase, cellobiose dehydrogenase, and chemical mediators: Combined actions for the decolorization of different classes of textile dyes

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 7003–7010

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: 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 2008 Available 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 the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes. Ó 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 are capable of degrading lignin and the other main wood components, fundamental for carbon flux in ecosystems. Their biodegradation capacities are due to highly non-specific, free-radical-mediated processes resulting from the activities of several enzymes secreted by these fungi such as laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) (Fu and Viraraghavan, 2001). These enzymatic systems enable white rot fungi to degrade a wide range of pollutants, including polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), pesticides, explosives, synthetic polymers and synthetic dyes (Pointing, 2001).

*

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

0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.01.019

Synthetic dyes are being increasingly used in the textile, paper, pharmaceutical, cosmetics and food industries. Over 7  105 tonnes of approximately 10,000 different dyes and pigments are produced annually worldwide, of which about 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 damaging the quality of the receiving streams and may be toxic to treatment processes, to food chain organisms and to aquatic life. For these reasons several countries are adopting stringent regulations for the release of colored industrial effluents. Azo, anthraquinone and indigo are the major chromophores found in commercial dyes. Decolorization of these dyes by physical or chemical methods is financially and often also methodologically demanding, time-consuming and mostly not very effective. Because of the range of chemical structures and properties, the degradation of mol-

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ecules of dyes in the environment by microorganisms is very slow (Pierce, 1994). Moreover, the industrially important azo dyes are cleaved under anaerobic conditions by bacterial azo-reductases to the corresponding amines, many of which are mutagenic and/or carcinogenic generating potential health hazards (Banat et al., 1996). At present, a number of studies have focused on the utilization of fungi since their mechanisms of dyes decolorization involve oxidative reactions which therefore do not produce toxic amines. However, using fungal biomass or single enzymes to remove color in a dye wastewater is still in the research stage. Recent studies have also shown that cellobiose dehydrogenase (CDH hereafter), an extracellular haemo-flavo-enzyme, produced by a number of wooddegrading and phytopathogenic fungi, has a role in the early events of lignocelluloses degradation and wood colonization (Henriksson et al., 2000), due to its ability to facilitate the formation of free hydroxyl radicals. CDH has been reported to display in vitro a synergism with laccases in the decolorization of an anthraquinonic dye, and directly in the oxidation of several chemicals (Vanhulle et al., 2007). Funalia (Trametes) trogii is a widely distributed white rot basidiomycete, good producer of laccases and other ligninolytic enzymes (Levin et al., 2005). A few studies on its capabilities in the decolorization of only some dyes have also been reported (Colao et al., 2006; Levin et al., 2001). The F. trogii strain 201, subject of this research, secretes laccases and no peroxidases under the different growth conditions utilized in this study and we here report for the first time that in the presence of cellulose besides laccases also CDH is secreted in abundance. In this paper, we report the investigation of the activities of the redox enzymes secreted by F. trogii 201 under different growth conditions on the decolorization of several classes of textile dyes among the most diffused in textile applications. The combined utilization of CDH or redox mediators with laccase was also investigated for the decolorization of the most recalcitrant dyes. 2. Methods 2.1. Chemicals All the chemicals were purchased from Sigma Chemical Co. Agar and Yeast Extract were from Oxoid Ltd. Textile dyes utilized were from Eurocolor S.p.A.; International Color 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) was maintained on basidiomycete rich medium (BRM) (Bezalel et al., 1997) agar plates at 4 °C and periodically transferred onto fresh BRM agar plates and grown at 28 °C. After 4–6

days of growth on agar plates, 500 ml shaken flask cultures containing 150 ml liquid BRM were prepared and inoculated with 10 plugs of fungal mycelia (about 25 mm2) and grown in the dark at 28 °C under continuous stirring at 130 rpm. After 4 days the grown mycelia were transferred in baffled 2000-ml Erlenmeyer flasks, closed with sterile air permeable silicon corks, containing 1000 ml of fresh BRM liquid medium and grown under the same conditions. The laccase expression was induced by the addition of 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 extracellular laccase activity reached a maximum about on day 7, the culture supernatant was collected by filtration through Whatman No. 1 paper and concentrated using an ultrafiltration Vivaflow 200 module (Sartorius group) with a 30,000 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 above described conditions but on modified BRM where 10 g l1 of microcrystalline cellulose powder was added as carbon source instead of glucose to obtain the simultaneous production of CDH and laccase. When the extracellular CDH activity reached a maximum, about on days 8– 9, the culture supernatant was collected by filtration and concentrated as reported above. 2.4. Enzyme assays Laccase activity was determined spectrophotometrically based on the capacity of this enzyme to oxidize the nonphenolic compound 2,20 -azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) (e420 = 36,000 M1 cm1), pH 3 at 25 °C; 1 U of laccase activity was defined as the amount of enzyme oxidizing 1 lmol substrate/min. CDH activity was assayed by following the decrease in absorbance of the electron acceptor, i.e. 2,6-dichlorophenol-indophenol (DCIP), at 520 nm (e520 = 6.8  103 M1 cm1), pH 4.0 and 37 °C. One unit of enzyme activity is defined as the amount of enzyme reducing 1 lmol DCIP/ min under the above reaction conditions. The combined determination of laccase and CDH activities was performed using 0.1 mM DCPIP following the method by Vasil’chenko et al. (2005). To understand if the DCPIP reducing activity observed was really due to CDH and not to a sugar oxidase usually present in the cellulolytic systems of fungi, i.e. glucose oxidase, in this assay we substituted cellobiose with D-glucose; no glucose oxidase activity was observed. MnP activity was estimated by the formation of Mn3+– tartrate complex (e238: 6500 M1 cm1) at pH 5, 25 °C. LiP activity was determined by the H2O2-dependent veratraldehyde (3,4-dimethoxybenzaldehyde) formation (e310 = 9300 M1 cm1), 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 extracellular fungal extract; then it was dialyzed against 10 mM sodium phosphate buffer (pH 6.0) and concentrated by ultrafiltration (membrane cut-off 30,000). All the following chromatographic steps were performed utilizing a HPLC Waters system composed by a 600 Solvent Module and a 996 diode array UV–vis detector interfaced to a personal computer running a Millennium chromatographic system. The laccase was then purified through the following sequential chromatographic steps: (1) DEAE macro prep ion exchange column (Bio-Rad, 2.6  14 cm), (2) PhenylSepharose 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 a A280/A600 ratio lower than 13, index of a purity larger than 99%, were pooled, concentrated and further used. 2.6. Enzymatic dyes decolorization The reaction mixtures for dyes-decolorizing activity were prepared in 50 ml shaken flasks and consisted of an aqueous solution of dye, 0.5 mg ml1 (0.5–1.1 mM) with the exceptions of Acid Blue 324: 0.3 mg ml1 (0.7 mM), Acid Red 374: 0.04 mg ml1(0.05 mM) and Acid Yellow 129: 0.08 mg ml1 (0.25 mM) (due to their lower water solubilities) in a total volume of 20 ml. The reactions were initiated adding crude extract or pure laccase and incubated at 30 °C with shaking (300 rpm) for the appropriate times. Samples of dye solutions were taken at regular times, centrifuged at 13,000 rpm for 5 min to eventually remove suspended particles and decolorization was measured after appropriate dilution. The dyes partially or non-decolorized by laccase were tested with the combined action of laccase and 4 mM 1-hydroxybenzotriazole (HBT) used as a redox mediator, the dye solution was adjusted to pH 5 using 0.1 M sodium citrate buffer. These dyes were also tested for decolorization with the combined action of laccase and CDH activities at pH 7. UV measurements were carried out on a double beam Perkin–Elmer EZ 301 spectrophotometer using 1 cm path length Hellma 110 quartz suprasil cells thermostated with a Lauda thermostat RE112. The UV/ vis absorption spectrum was recorded (300–800 nm) for each dye and decolorization was followed monitoring the absorption 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 maximum on days 7–9 of cultivation. It was stimulated by the addition of CuSO4 or veratryl alcohol to the growth medium.

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Activity increases from 2.0 U ml1 to 4.0, 6.0 or 7.5 U ml1 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 of 0.25 mM veratryl alcohol (instead of Cu(II)) after 2 days of growth resulted in an increase of laccase production to 7.2 U ml1, comparable to 7.5 U ml1 obtained with the supplement of 1.0 mM CuSO4. Detectable CDH production occurred only in media containing cellulose powder as carbon source. For such purpose the BRM medium was modified by substituting glucose, which inhibits CDH expression, with 10 g l1 cellulose (Stapleton and Dobson, 2003). The best CDH production was obtained on days 8–9 of cultivation (up to 0.6 U ml1). At the same time the laccase activity reached about 5–6 U ml1. MnP and LiP activities were not detected in any of the different conditions 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 their extensive utilization in dyeing factories. Several mono-azo (Acid Yellow 49, Acid Red 42, Reactive Yellow 39, Reactive Red 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 anthraquinonic dyes (Reactive Blue 69, Acid Blue 80, Acid Blue 324) were tested for decolorization utilizing a crude extracellular culture extract of F. trogii containing laccase activity (1.5 U ml1). The time courses for the dyes decolorizations were followed for at least 3 days but the maximal effect was substantially reached within 24 h for all the dyes tested. The initial experiments were performed at pH 3.0 since the main laccase from F. trogii showed the maximal activity at such pH value (Garzillo et al., 2001). As shown in Table 1, at such pH only the dyes which had adequate water solubility were investigated. The chromo-complexed azo Acid Blue 158, Acid Black 194, Acid Red 186, the disazo Acid Black 1, and the anthraquinonic Reactive Blue 69 were decolorized to a large extent (>85%) (see Table 1), whereas for the monoazo Reactive Yellow 39, Reactive Red 272, and the disazo Direct Red 243 only 17–28% decolorization was achieved. All the dyes were subsequently tested at pH 7.0 where they were all soluble. In particular, the dyes insoluble at pH 3 gave the following results: the mono-azo Acid Red 42, the disazo Acid Red 374, the anthraquinonic Acid Blue 324 and Acid Blue 80 were largely decolorized (see Table 1) but the mono-azo Acid Yellow 49, the mono-azo chromo complexes Acid Yellow 129, the tri-azo Direct Blue 71, and the poly-azo Direct Black 22 resulted in a lower decolorization yield. Regarding the dyes tested at both pH 3 and 7, we noted comparable or even better decolorization results at pH 7.0 using the same amount of laccase, although the main laccase from F. trogii shows about 10 times reduced activity towards ABTS or DMP at that pH

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Table 1 Decolorization (%) 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 Acid Red 42 Reactive Yellow 39

– – 21.0

23.3 93.4 0.0

35.4 n.d. 81.6

21.4

15.9

79.5

96.3 (26.3)a n.d. 87 (74.2)a, pH 3 0.0, pH 7 69 (27)a

Reactive Red 272 Monoazo chromo complexes

Acid Acid Acid Acid

82.4 99.0 – 96.6

99.3 88.7 27.5 96.5

n.d. n.d. 23.5 n.d.

n.d. n.d. 70 (35)a n.d.

Disazo

Acid Black 1 Direct Red 243 Acid Red 374

85.0 28.2 –

95.0 90.4 100.0

n.d. 92.0 n.d.

n.d. n.d. n.d.

Tri-azo Poly-azo

Direct Blue 71 Direct Black 22

– –

21.5 31.3

91.5 64.3

97.4 (32.5)a 100.0 (42)a

Anthraquinonic

Reactive Blue 69 Acid Blue 80 Acid Blue 324

96.7 – –

91.1 98.6 75.0

n.d. n.d. 85.0

100.0 (100)a n.d. 95 (75)a

Blue 158 Black 194 Yellow 129 Red 186

a

The data in parenthesis are the results of the decolorization experiments performed utilizing the fungal extract containing CDH plus laccase activities without the addition of lactose. No quantification of the oligosaccharides content was performed on such extracts.

3.3. Dyes decolorization with the addition of HBT mediator All the selected dyes which were not decolorized to a large degree utilizing the sole crude fungal extract were then subjected to tests employing the synthetic mediator HBT, one of the best radical mediators for the laccase sys-

A Absorbance (538 nm)

1.6 1.4 1.2 1.0

pH 3.0

0.8 0.6 0.4

pH 7.0

0.2 0.0 0

B

10

20

30

40

50

1.0

Absorbance (515 nm)

(Garzillo et al., 2001). The largest difference was observed for the disazo Direct Red 243 which showed a three times increase of decolorization at pH 7.0 (pH 3.0: 28.2%, pH 7.0: 90.4%) (Fig. 1A). The effect of decreasing the amount of starting laccase activity was tested on a chromo-complexed mono-azo dye, Acid Black 194: reducing the laccase amount from 1.5 to 1.0 U ml1 did not substantially change the extent (99%) and the course of decolorization; further reductions to 0.75 and 0.375 U ml1 resulted in decreased levels of decolorization (91% and 84%, respectively, at 24 h). Decolorizations by purified laccase and crude extracellular fungal extract were also compared. Acid Black 194 was completely decolorized by the crude extract, whereas the application of the same amount of pure laccase resulted in a 77% decolorization only. The anthraquinonic Reactive Blue 69 was instead decolorized by the same extent with both pure enzyme and crude extract (95%). All the dyes totally or partially decolorized by the catalytic action of extract or pure laccase in the course of the present study did produce extensive precipitation of polymerized products, within 24 h, easily eliminated by decanting, filtering or through low speed centrifugation. Furthermore, as previously suggested, laccase oxidation might detoxify azo dyes because this reaction releases azo linkages as molecular nitrogen, impeding toxic aromatic amine formation (Chivukula and Renganathan, 1995).

0.8 0.6 0.4 0.2 0.0 0

10

20

30

40

50

60

Time (hours) Fig. 1. (A) Effect of pH on the decolorization of Direct Red 243 utilizing crude 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 values of three replicates are shown. (B) Time course of the decolorization of Reactive Red 272 obtained with the addition of crude extracellular culture extract of F. trogii: containing laccase (d) and laccase plus CDH and 30 mM lactose (.) activities, pH 7.0, 30 °C. Control (s). Mean and standard deviation values of three replicates are shown.

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

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HBT is a stronger oxidant than laccase itself (redox potential of F. trogii laccase 760 mV at pH 7.0, and of radical HBT: 1084 mV) (Garzillo et al., 2001; Zille et al., 2004). The mono-azo dyes Reactive Yellow 39 and Reactive Red 272, the tri-azo Direct Blue 71, the poly-azo Direct Black 22, recalcitrant to the sole action of laccase, and the anthraquinonic dye Acid Blue 324 subjected to the action of the laccase/HBT system, resulted in an extensive decolorization within 4 h. On the contrary the mono-azo Acid Yellow 49 and the mono-azo chromo-complex Acid Yellow 129 were substantially decolorized to the same extent 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 expensive chemical mediators the action of laccase was combined to that of CDH, an enzyme able to indirectly generate in a Fenton type reaction hydroxyl radicals, very potent oxidants. CDH was expressed by F. trogii only when grown on media containing cellulose and no glucose, as reported in Section 2. In this medium the fungus secreted CDH but also laccase activities. The decolorizing action of crude extracellular culture extracts containing CDH (0.45 U ml1) and laccase (1.5 U ml1) activities was compared to that of extracts containing laccase activity only (1.5 U ml1). The experiments were performed at pH 7.0 with the exception of Reactive Yellow 39 which was tested both at pH 7.0 and 3.0. Furthermore, since the action of CDH is associated to the oxidation of cellobiose, lactose, or similar carbohydrates, decolorization experiments with extracts containing CDH were performed in the absence or in the presence of 30 mM lactose. This carbohydrate was essential to activate the CDH to the production of hydrogen peroxide which further generates, in a Fenton type reaction, hydroxyl radicals (see Section 4). The presence of CDH plus lactose results in an improved reduction of color intensity for all the dyes tested, much higher than with laccase activity alone (Table 1). In Fig. 1B is shown the time course of the decolorization of Reactive Red 272 obtained with the addition of crude extracellular culture extract of F. trogii containing laccase alone and laccase plus CDH and 30 mM lactose. The addition of CDH yields a 4.3 times improvement in Reactive Red 272 decolorization. Also the anthraquinonic dyes Reactive Blue 69 and Acid Blue 324 were decolorized to a higher extent when CDH was utilized. Experiments performed adding lower concentrations of lactose (3 mM) or the CDH extract without lactose resulted in improvements in dyes decolorization but to lower extents with respect to CDH plus 30 mM lactose (see Table 1). 4. Discussion 4.1. Enzymes induction White rot basidiomycetes, involved in wood decay worldwide, and among them several different strains of F.

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trogii have been shown to express several ligninolytic enzymes mainly laccases as well as lignin and manganese peroxidases (Levin et al., 2002). Under the conditions described here, F. trogii 201 produces a major phenol oxidase (Colao et al., 2006; Garzillo et al., 1998, 2001); it does not produce any observable peroxidase activity. Induction of laccase activity utilizing copper ions or lignin derived aromatic compounds such as veratryl alcohol allowed more than a three-fold increase in laccase activity (Levin et al., 2002; Palmieri et al., 2003). CDH, another enzyme thought to be involved in lignocellulose degradation, is detected in F. trogii 201 when glucose is absent from the cultural medium and substituted by cellulose as previously observed for some white rot, brown rot and soft rot fungi (Stapleton and Dobson, 2003). 4.2. Dyes structure and biodegradability using laccase The decolorization of various dyes with different structural patterns was investigated using crude extracellular culture extract and pure laccase from F. trogii. Our system was able to efficiently degrade a number of commercial textile dyes at pH 3.0. The decolorization of the antraquinonic dyes by laccase was expected, since high potential laccases have been shown to decolorize anthraquinonic dyes more efficiently than other classes of dyes (Champagne and Ramsay, 2005). Nevertheless, the most employed dyes belong to the azo class which accounts for the 70% of all textile dyes produced. It has been observed that laccases display substrate specificities and the chemical structures of the dyes mainly due to differences in electron distribution, charge density as well as steric hindrances largely influence their decolorization extent and rates (Chivukula and Renganathan, 1995; Pasti-Grigsby et al., 1992). Laccases modify azo dye structures by destroying their chromophoric assemblies, phenoxyl radicals are generated in the reaction course (Chivukula and Renganathan, 1995). In a first step, one electron is abstracted from the phenolic/naphtholic ring, yielding a phenoxy radical; the abstraction of a second electron, generates an aromatic cation which can be stabilized by electron-donating groups present in the ring. Actually the best biochemical decolorizations were previously achieved with those azo dyes that carried hydroxyl groups, strong electron donating moieties, in ortho and para positions to the azo bond (Kandelbauer et al., 2004). The meta-substituted analogues were not attacked by laccase alone since the activating strength is lower in such position. Electron withdrawing substituents such as halogen or nitro groups on the aromatic rings, make it difficult for oxidases to form cation radicals thus inhibiting dyes degradation. Instead, azo dyes characterized by even weakly electron-donating methyl groups were decolorized efficiently (Pasti-Grigsby et al., 1992). Furthermore, heterocyclic azo dyes, containing pyrazole or triazole rings, were not decolorized significantly unless were present hydroxyl and other electron donating groups on the heterocyclic and vicinal aromatic rings, in ortho position to the

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azo bond. Other effects, such as those caused by reaction intermediates, may contribute as well (Kandelbauer et al., 2004). In the present study, all the mono-azo chromo-complexed dyes tested at pH 3.0 were extensively decolorized (Acid Blue 158, Acid Black 194, and Acid Red 186) since the oxidation was promoted by the hydroxyl groups in ortho positions to the azo bonds on the vicinal rings. Instead, the mono-azo dyes checked at pH 3.0 (Reactive Yellow 39 and Reactive Red 272) resulted to be recalcitrant to decolorization because heterocyclic rings with chlorine atoms on them or in the surrounding aromatic rings as well as sulfonic groups in ortho position to the azo were present. Regarding the two disazo dyes examined at pH 3.0 Acid Black 1 was 85% decolorized, whereas Direct Red 243 was degraded to a lower extent (Table 1) for the presence of a central heterocyclic pyrazole ring and a higher number of sulfonic groups in ortho to the azo bond in Direct Red 243. The crude extracellular culture extract of F. trogii utilized at pH 7.0 yielded high decolorization of all the anthraquinonic dyes tested (Reactive Blue 69, Acid Blue 324 and Acid Blue 80). Among the mono-azo dyes tested only Acid Red 42 was largely decolorized, whereas Acid Yellow 49, Reactive Yellow 39 and Reactive Red 272 were mostly recalcitrant to degradation due to the presence of electron-withdrawing halogen atoms and sulfonic groups in ortho positions to the azo bonds and heterocyclic rings, whereas in the Acid Red 42 a hydroxide and an amino group activate the azo bond attack. Regarding the mono azo chromo complexes the Acid Blue 158, Acid Black 194 and Acid Red 186 were all degraded to large extents due to two activating hydroxyl groups in ortho to the azo bonds either in homo- or hetero-cyclic rings; absent in the poorly decolorized Acid Yellow 129 (Table 1) which also carries a deactivating carboxyl group. All the disazo dyes tested in our experiments were decolorized at pH 7.0 since they showed at least one hydroxyl group in ortho position with respect to the azo bond. The Acid Black 1 and particularly the Direct Red 243 were better decolorized at pH 7.0, than at pH 3.0 (Fig. 1A). On the contrary the triazo Direct Blue 71, and the poly-azo Direct Black 22 were scarcely decolorized probably due to the higher number of azo bonds surrounded by scarcely activated aromatic rings and also to steric factors due to the large hindrances of these two molecules (Table 1). 4.3. Redox potential and biodegradability using laccase All the structural differences mentioned above result in substantial electron distribution and charge density variations influencing the redox potential of the dyes. A correlation between the enzyme redox potential and its activity toward substrates has been described (Xu et al., 1998) and the driving force for the redox reaction catalyzed by laccases is expected to be proportional to the difference between the redox potentials of the oxidizing enzyme and

the reducing substrate (dye). A lower Eo of substrate and/or a higher Eo of laccase normally results in a higher rate of substrate oxidation and a linear correlation between the percentage decolorization of each dye and the respective redox potential was found (Zille et al., 2004). The redox potential of phenolic or naphtholic substrates decreases when pH increases since the mechanism of oxidative proton release is favored at high pH. According to Xu, a phenolic compound shows a decrease of Eo equal to 0.059 V for each pH unit increased (at 25 °C), therefore a pH change from 3 to 7 would result in an Eo (phenol) decrease of about 0.24 V (Xu, 1997). Over the same pH range, the Eo change for laccases is generally much smaller (about 0.03 V for the main laccase from F. trogii) (Garzillo et al., 2001). Such pH dependences would then result in a larger difference in redox potential between phenolic substrates and laccase at higher pH, leading to an activity increase for phenols oxidation as the pH increases (Xu, 1997). This explains why the dyes tested at both pH 3 and 7 are usually decolorized at better levels at pH 7.0: the disazo Direct Red 243 presents a three times increase of decolorization at pH 7.0 with respect to pH 3.0 (Fig. 1A). Also the disazo dye Acid Black 1 and the mono azo chromo-complexed Acid Blue 158 were better decolorized at pH 7.0. All of them exhibit phenolic/naphtholic rings directly connected to the azo moieties. Contrarily to what generally observed in the literature our laccase system has been able to decolorize not only the anthraquinonic dyes but also a substantial part of the azo dyes tested. 4.4. Further observations It has been previously noticed that over long periods of oxidation, there can be a coupling between the reaction products, and even polymerization. It is known that laccases can catalyze the polymerization of various halogen-, alkyl-, and alkoxy-substituted anilines as well as phenolic, naphtholic, and aminophenolic compounds (Aktas and Tanyolac, 2003). Contrarily to what observed in the investigation by Zille et al. where soluble polymerized products provide unacceptable color levels in effluents (Zille et al., 2005), in all the decolorization performed in the present study, polymerization generated substantial precipitation of the dyes within 24 h. A comparison between the action of purified laccase and of crude extracellular extract showed that, whereas the crude extract completely decolorized Acid Black 194; pure laccase application resulted in only 77% decolorization; possibly the presence of natural mediators in the extracellular fungal culture broth favours dye decolorization. 4.5. Decolorizations utilizing the laccase/HBT combination Those dyes that were scarcely decolorized by laccase alone were subjected to the combined action of laccase and HBT mediator which was previously shown to be capable to extend the oxidation power of laccases. Laccases

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oxidize redox mediators forming short-lived cation radicals, which co-oxidize the substrate. These cation radicals can be formed by two mechanisms: the redox mediator can perform either a one-electron oxidation of the substrate (Bourbonnais et al., 1998) or it extracts an H-atom from the substrate (Fabbrini et al., 2002). HBT operates according to the second mechanism and the free-radical produced is an oxidant stronger than laccase itself (+1.084 V) (Reyes et al., 1999). Thus, the laccase/mediator system is able to decolorize dyes having higher redox potentials than laccase alone (Claus et al., 2002). Among the dyes tested in the present study all the molecules recalcitrant to decolorization by laccase alone were degraded with HBT, only exceptions Acid Yellow 49 and Acid Yellow 129 (see Table 1). Probably the presence on both dyes of heterocyclic (pyrazole) rings flanking the azo bond on one side and on the other side aromatic rings containing only electron withdrawing substituents make these dyes resistant even to the mediator attack. 4.6. Decolorizations utilizing the laccase/CDH combination CDH is an extracellular fungal flavocytochrome enzyme secreted by several wood degrading fungi. While its physiological function is not clearly known, it is capable to preferentially oxidize oligosaccarides like cellobiose, cellotriose, or lactose to the corresponding lactones using a wide spectrum of electron acceptors; among them the reduction 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). The attack of hydroxyl radicals generated by CDH can yield the demethoxylation and/or hydroxylation of many aromatic compounds, possibly leading to the conversion of nonphenolic structures to phenolic ones, thus rendering the molecule easily oxidized by laccases or peroxidases (Hilden et al., 2000). In the present investigation, we observed enhancements in the decolorization of dyes recalcitrant to laccase due to the further addition of CDH and lactose; the latter needed as the CDH reducing substrate. When we utilized the crude extract from culture broths modified for the production of CDH and thus initially containing cellulose we also obtained decolorization enhancements without the addition of lactose. This could be due to the presence of cellulose derived cellobiose or other CDH reducing substrates in the extract. Even in the case of hydroxyl radicals produced by CDH the reaction rate has been reported to depend on the basic structure of the molecule and on the nature of auxiliary groups attached to the aromatic nuclei of the dyes (Galindo and Kalt, 1999). In the present study, the addition of CDH resulted in substantial increases in decolorization for all the dyes recalcitrant to the action of laccase alone. The combined action of laccase and CDH activated by lactose always yielded decolorization values above 69%.

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We can therefore conclude that raw fungal extracts could be utilized in place of more expensive chemical treatments and synthetic mediators; in particular if the action of laccase is supported by the formation of hydroxyl radicals triggered by enzymes such as cellobiose dehydrogenase. Acknowledgements We thank the Assessorato all’Istruzione, Formazione e Lavoro, Regione Toscana and we gratefully acknowledge the support of Progetto MECHOS, POR Ob. 3 2000/ 2006 Toscana, Progetti integrati di ricerca Mis. D4 (Decreto Regionale 03/04/2007 no. 1785). References Aktas, N., Tanyolac, A., 2003. Reaction conditions for laccase catalyzed polymerization of catechol. Bioresour. Technol. 87, 209–214. Banat, I.M., Nigam, P., Singh, D., Marchant, R., 1996. Microbial decolorization of textile-dye-containing effluents: a review. Bioresour. Technol. 58, 217–227. Bezalel, L., Hadar, Y., Cerniglia, C.E., 1997. Enzymatic mechanisms involved in phenanthrene degradation by the white rot fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 63, 2495–2501. Bourbonnais, R., Leech, D., Paice, M.G., 1998. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta 1379, 381–390. Cameron, M.D., Aust, S.D., 2001. Cellobiose dehydrogenase – an extracellular fungal flavocytochrome. Enzyme Microb. Technol. 28, 129–138. Champagne, P.P., Ramsay, J.A., 2005. Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor. Appl. Microbiol. Biotechnol. 69, 276–285. Chivukula, M., Renganathan, V., 1995. Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl. Environ. Microbiol. 61, 4374– 4377. Claus, H., Faber, G., Konig, H., 2002. Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl. Microbiol. Biotechnol. 59, 672–678. Colao, M.C., Lupino, S., Garzillo, A.M., Buonocore, V., Ruzzi, M., 2006. Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb. Cell Fact. 5, 31. Fabbrini, M., Galli, C., Gentili, P., 2002. Radical or electron-transfer mechanism of oxidation with some laccase/mediator systems. J. Mol. Catal. B: Enzym. 18, 169–171. Fu, Y., Viraraghavan, T., 2001. Fungal decolorization of dye wastewaters: a review. Bioresour. Technol. 79, 251–262. Galindo, C., Kalt, A., 1999. UV/H2O2 oxidation of azodyes in aqueous media: evidence of a structure–degradability relationship. Dye Pigment 42, 199–207. Garzillo, A.M., Colao, M.C., Buonocore, V., Oliva, R., Falcigno, L., Saviano, M., Santoro, A.M., Zappala, R., Bonomo, R.P., Bianco, C., Giardina, P., Palmieri, G., Sannia, G., 2001. Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii. J. Protein Chem. 20, 191–201. Garzillo, A.M., Colao, M.C., Caruso, C., Caporale, C., Celletti, D., Buonocore, V., 1998. Laccase from the white-rot fungus Trametes trogii. Appl. Microbiol. Biotechnol. 49, 545–551. Henriksson, G., Johansson, G., Pettersson, G., 2000. A critical review of cellobiose dehydrogenases. J. Biotechnol. 78, 93–113. Hilden, L., Johansson, G., Pettersson, G., Li, J., Ljungquist, P., Henriksson, G., 2000. Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation? FEBS Lett. 477, 79–83.

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