Enzyme and Microbial Technology 40 (2007) 16731677

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by puried laccase and Mn-peroxidase
Vanja Kokol a , Ale Dolika a , Ivana Eichlerov b , Petr Baldrian b, , Frantiek Nerud b s s a s
b

Institute of Textiles, University of Maribor, Maribor, Slovenia Laboratory of Biochemistry of Wood-Rotting Fungi, Institute of Microbiology ASCR, Vde sk 1083, CZ 14220, Praha 4, Czech Republic n a Received 19 June 2006; received in revised form 30 July 2006; accepted 12 August 2006

Abstract Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L1 ), the MnP on day 14 (34.5 U L1 ). Laccase and Mn-peroxidase were puried from the culture liquid using gel permeation and ion-exchange chromatographies. Puried Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (34). 2006 Elsevier Inc. All rights reserved.
Keywords: Ischnoderma resinosum; Basidiomycetes; Laccase; Mn-peroxidase; Textile dyes; Ligninolytic enzymes

1. Introduction Wastewaters from textile industries represents a serious problem all over the world. They contain different types of synthetic dyes, which are mostly toxic, mutagenic and carcinogenic. Moreover, they are very stable to light, temperature and microbial attack, making them recalcitrant compounds. Conventional wastewater treatment systems are often inefcient and existing physical and chemical technologies are expensive, time-consuming and methodologically demanding. Currently, one of the possible alternatives for treatment of textile efuents is the use of ligninolytic fungi, which can oxidize a wide spectrum of organic pollutants including synthetic dyes [13]. Biodegradative ability of white-rot fungi is generally assumed to be associated with the production of extracellular ligninolytic enzymes [4,5], among them laccase and manganese peroxidase being the most intensively studied. As the use of whole fungal cultures for decolorization is not easily applicable in a large scale [6], at present, the studies of in vitro decoloriza-

Corresponding author. Tel.: +420 241062315; fax: +420 241062384. E-mail address: baldrian@[Link] (P. Baldrian).

tion of synthetic dyes by crude or puried enzymes becomes more important [711]. The phenol oxidase laccase (p-diphenol: oxygen oxidoreductase; EC [Link]) has been studied for a long time [1214]. It is a multicopper enzyme which catalyses the oxidation of wide number of phenolic compounds and aromatic amines but its substrate specicity can be extended to non-phenolic compound in the presence of low molecular mass compounds acting as mediators [15,16]. Manganese peroxidase (EC [Link]) is a heme-containing enzyme catalysing the oxidation of Mn2+ to Mn3+ , which in turn can oxidise a variety of phenolic substrates [17,18]. Many white-rot fungi (e.g. Phanerochaete chrysosporium, Pleurotus ostreatus, Bjerkandera adusta, Trametes versicolor, etc.) and their enzymes have been intensively tested in connection with the decolorization of synthetic dyes [9,1924]. Recently, we demonstrated both high production of ligninolytic enzymes and decolorization of synthetic dyes Orange G and Remazol Brilliant Blue R by liquid cultures of the white-rot fungus Ischnoderma resinosum [25]. The main objectives of this work were: (i) to evaluate the contribution of the ligninolytic enzymes laccase and manganese peroxidase of I. resinosum to the degradation of synthetic dyes, commonly used in the textile industry and (ii) to test the possible enhancement of decolorization by the use of laccase redox mediators and the ability of I.

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/[Link].2006.08.015

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resinosum to decolorize textile dyes contained in the synthetic dyebaths.

2. Material and methods 2.1. Organism and cultivation
I. resinosum (Fr.) P. Karst CCBAS 553 was obtained from the CCBAS collection (Institute of Microbiology ASCR, Prague, Czech Republic). The strain was maintained by serial transfers and kept on wort agar slants at 4 C. Cultivation was carried out in 100 mL Erlenmeyer asks with 20 mL of Nlimited (0.2 g L1 of ammonium tartrate) liquid Kirk medium [26]. The asks were inoculated with two wort agar plugs (2 C balling, 10 mm diameter), cut from the actively growing part of a colony on a Petri dish, and incubated at 27 C for 20 days. Enzyme activity was measured in culture ltrates detained after mycelia removal. Biomass production in liquid media was evaluated by determining the dry mass of mycelia. Mycelia were harvested from the cultivation asks, washed with distilled water, dried at 105 C for 24 h and weighed.

2.4. Decolorization
Synthetic dyes Reactive Black 5 (RK; azo), Reactive Blue 19 (RB; anthraquinone), Reactive Red 22 (RR; azo), and Reactive Yellow 15 (RY; azo) were used at a concentration of 100 ppm in all experiments. Decolorization was expressed as per cent decrease of absorbance at the respective absorbance maxima: 595 nm for RK, 592 for RB, 519 for RR, and 416 for RY. The decolorization reactions included: (a) puried laccase from I. resinosum (25 mU/mL), with or without the redox mediators violuric acid (VA, 5 mM) and hydroxybenzotriazole (HBT, 5 mM); (b) puried Mn-peroxidase from I. resinosum (115 mU/mL) with 10 mM H2 O2 and 20 mM MnSO4 . 100 mM succinatelactate buffer, pH 4.5 was used to provide the organic acid for Mn2+ chelation; (c) culture liquid from I. resinosum culture diluted with distilled water to contain 25 mU/mL laccase and 115 mU/mL Mn-peroxidase, with or without 5 mM VA, 5 mM HBT, or 10 mM H2 O2 and 20 mM MnSO4 in 100 mM succinatelactate buffer, pH 4.5. The effect of pH on dyes decolorization was examined in the pH range 3.08.0 in a 0.1 M citrate0.2 M phosphate buffer using culture liquid from I. resinosum culture diluted with distilled water to contain 25 mU/mL laccase and 115 mU/mL Mn-peroxidase, with or without 5 mM VA, 5 mM HBT, or 10 mM H2 O2 and 20 mM MnSO4 . In the same experiment, culture liquid was also used for the decolorization of dyes dissolved in water (initial pH around 6.1) and within synthetic dyebaths (initial pH around 5.7). The dyebaths contained (per liter): 1200 mg NaCl, 5 mg Na2 CO3 , 5 mg NaOH, 10 mg EDTA, and 10 mM acetic acid. All decolorization reactions were run in three replicates and proceeded at 25 C in the dark.

2.2. Ligninolytic enzyme assays

Activities of extracellular laccase (EC [Link]) and manganese peroxidase (EC [Link], MnP) were determined spectrophotometrically by monitoring the absorbance increase at 425 nm (laccase) or 590 nm (MnP) in the reaction mixture. Laccase activity was assayed according to [27] by monitoring the oxidation of ABTS. Determination of MnP activity using MBTH and DMAB was based on the method of [28] modied according to [29]. MBTH and DMAB were oxidatively coupled by the action of the enzyme in the presence of added H2 O2 and Mn2+ ions to give a purple indamine dye product. The values were corrected for the activities in the test samples (a non-specic peroxidase activity) without manganese, where manganese sulfate was substitued by ethylenediaminetetraacetate (EDTA) to chelate Mn2+ ions present in the extract. All measurements were repeated three times. One unit of enzyme activity (U) was dened as an amount catalyzing the production of 1 mol of green or purple product per mL per min.

3. Results Under our experimental conditions, I. resinosum produced extracellular ligninolytic enzymes laccase and MnP, lignin peroxidase was not detected (data not shown). The activity of laccase achieved the maximum on day 10 (29.4 U L1 ), the MnP on day 14 (34.5 U L1 ) (Fig. 1). The biomass production was 45 g L1 with the maximum on day 14. Laccase and Mn-peroxidase were puried from the culture liquid using gel permeation and ion-exchange chromatographies. The decolorization of 100 ppm solutions of all textile dyes tested in this study with puried laccase (25 mU/mL) was detected only with PB and PY, the dyes PB and PR were not

2.3. Purication of laccase and MnP

The culture liquid from a 30-day culture of I. resinosum grown on N-limited Kirk medium [26] was ltered and concentrated by ultraltration. The concentrate was loaded onto a DEAEsepharose column (Pharmacia LKB, HR 10/10) equilibrated with 20 mM phosphate buffer, pH 6.0. The proteins were eluted with a gradient of 01 M NaCl at a ow rate of 0.5 mL min1 . The active fractions were pooled, desalted and applied to a CIM QA Disk (BIA Separation, Slovenia) equilibrated with 20 mM phosphate buffer, pH 6.0. The fractions were eluted with a gradient of 01 M NaCl at a ow rate of 4 mL min1 . Desalted active fractions were applied again to CIM QA Disk (BIA Separation, Slovenia) equilibrated with 20 mM phosphate buffer, pH 7.0 and the proteins were eluted under the conditions described above. After this step the concentrated puried laccase fractions were pooled and kept frozen at 18 C. MnP purication process continued with two other steps, where active fractions were desalted and again chromatographed on CIM QA Disk (BIA Separation, Slovenia) equilibrated with 20 mM phosphate buffer, pH 7.0. The proteins were eluted with a gradient of 01 M NaCl at a ow rate of 4 mL min1 . In the last step the MnP fractions were desalted, chromatographed on CIM QA Disk (BIA Separation, Slovenia), equilibrated with 50 mM acetate buffer, pH 4.5, and the proteins were eluted under the conditions described above. Concentrated puried MnP fractions were pooled and kept frozen at 18 C. The enzymes were puried until electrophoretic homogeneity (checked by SDS-PAGE). The effect of pH on laccase and MnP activities was examined in the pH range 3.08.0 in a 0.1 M citrate0.2 M phosphate buffer at 25 C. Small molecular mass fraction of culture liquid was obtained by ultraltration using Amicon stirred cell (Millipore, USA) with a 10 kDa cut-off membrane. The small molecular mass fraction did not contain detectable laccase activity.

Fig. 1. Activity of ligninolytic enzymes and production of biomass by I. resinosum in liquid N-limited medium. The data represent averages and standard deviations (n = 3).

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Fig. 2. Decolorization of 100 ppm of the dyes: Reactive Black 5 (a), Reactive Blue 19 (b), Reactive Red 22 (c), and Reactive Yellow 15 (d) after a 1 and 24 h incubation with puried laccase or Mn-peroxidase and with the culture liquid (CL) of I. resinosum combined with violuric acid (VA), hydroxybenzotriazole (HBT), or Mn and H2 O2 . The data represent averages and standard deviations (n = 3).

decolorized. MnP (115 mU/mL) decolorized all textile dyes, but the decolorization was low (Fig. 2). The treatment of dyes with the culture liquid from I. resinosum cultures containing the same total amount of ligninolytic enzymes (25 mU/mL laccase and 115 mU/mL MnP) resulted generally in higher decolorization than the treatment using individual enzymes. Interestingly, this was independent on the presence of Mn and H2 O2 indicating the insignicant involvement of MnP in the decolorization by total culture liquid. The decolorization was signicantly improved by the addition of laccase synthetic mediators violuric acid and hydroxybenzotriazole. These compounds increased the decolorization to 8090% after a 24 h treatment. In the case of RK and RY decolorization around 80% was already achieved after 1 h incubation (Fig. 2). Higher decolorization rates were recorded using VA. The decolorization of synthetic dyebaths, i.e. the synthetic mixtures imitating the efuents of textile dyeing processes was slightly less efcient than for dyes dissolved in water with the exception of the treatment of RR-containing dyebath in the absence of redox mediators (Fig. 3). The decolorization of dyes was strictly dependent of the pH of the treated solution. The highest decolorization rates were recorded at pH 45, while the decolorization at pH 78 only occurred in the presence of HBT (Table 1). The pH optima for both ligninolytic enzymes were detected at pH 5, but both laccase and MnP also exhibited reasonable activity at pH 6. The pH of

synthetic dye solutions in water was around six while it was 5.5 in the synthetic dyebaths. To test the possible contribution of small molecular mass molecules on laccase activity as possible laccase activators or redox mediators, dye decolorization was also tested with puried laccase supplemented with a small molecular mass fraction

Fig. 3. Decolorization of dyes (100 ppm) in water (black bars) and in synthetic dyebath (dashed bars) after 24 h incubation with the culture liquid (CL) of I. resinosum combined with violuric acid (VA), hydroxybenzotriazole (HBT), or Mn and H2 O2 . The data represent averages and standard deviations (n = 3).

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Table 1 Activity of puried laccase and Mn-peroxidase and decolorization of dyes after 24 h incubation with culture liquid (CL) of I. resinosum combined with violuric acid (VA), hydroxybenzotriazole (HBT), or Mn and H2 O2 in 0.1 M citrate and 0.2 M phosphate buffer, pH 38 pH 3.0 Laccase Mn-peroxidase Reactive Black 5 CL CL + VA CL + HBT CL + Mn + H2 O2 Reactive Blue 19 CL CL + VA CL + HBT CL + Mn + H2 O2 Reactive Red 22 CL CL + VA CL + HBT CL + Mn + H2 O2 19 8 100 39 100 100 100 75 100 100 100 85 100 100 4.0 57 75 77 100 84 81 58 100 67 89 55 100 55 89 5.0 100 100 34 50 50 67 4 0 57 88 0 19 22 67 6.0 49 79 16 39 46 0 0 0 55 0 0 0 0 0 7.0 5 53 0 0 37 0 0 0 29 0 0 0 0 0 8.0 4 15 0 0 28 0 0 0 19 0 0 0 0 0

Data are expressed per cent of the highest value.

(<10 kDa) of the culture liquid. No signicant increase of activity was observed in any case (results not shown). 4. Discussion It was demonstrated, that I. resinosum cultures are able to efciently decolorize synthetic dyes Orange G and Remazol Brilliant Blue R [25], however, the contribution of ligninolytic enzymes to this process was not clear. It has been reported, that puried laccase is able to decolorize several synthetic dyes [3,30]. However, the substrate specicity of laccase enzymes of different origin varies considerably [3133]. The puried laccase from I. resinosum was able to decolorize Reactive Blue 19 that was also decolorized by laccases from Pycnoporus cinnabarinus, Trametes villosa and Coriolopsis gallica [31,34], but it was unable to decolorize Reactive Black 5, the dye transformed by laccases from Daedalea quercina and T. versicolor [33,35]. It is highly probable that the structure and redox potentials of individual laccases and dyes are responsible for these differences in substrate specicity. The addition of redox mediators thus leads to the decolorization of a broader spectrum of synthetic dyes or to the increase in decolorization rates [31,34,36]. In this study, violuric acid was found to be a more efcient redox mediator than hydroxybenzotriazole, which is in agreement with previous reports [37,38]. Although puried Mnperoxidases from T. versicolor was demonstrated to efciently decolorize the synthetic dye Reactive Black 5, the decolorization rate with the puried I. resinosum enzyme was low. Although dye decolorization with fungal cultures and puried ligninolytic enzymes attracted considerable attention, the relative contribution of different enzymes in fungal cultures

to decolorization was not yet completely understood. It was demonstrated, that a strain of P. ostreatus with high laccase production performed faster decolorization than the strain with low laccase production [39]. The comparison of culture liquid from I. resinosum and individual puried enzymes demonstrated, that the activity of culture liquid is higher than the activity of the same amount of isolated enzymes. This cannot be explained by the production of small molecular mass redox mediators of laccase activity since there was no effect of small molecules in the culture liquid on laccase decolorization activity. Moreover, it seems that the role of Mn-peroxidase in decolorization is negligible since the addition of Mn and H2 O2 to the culture liquid did not increase decolorization. The use of redox mediators increased signicantly the decolorization rates of all tested dyes not only when combined with puried laccase, but also in the combination with I. resinosum culture liquid. This is mainly important from the technological viewpoint since the culture liquid can be used for decolorization without the need of enzyme purication steps. The limitation for the use of I. resinosum cultures for dye decolorization is the acidic pH range of activity similar to other white-rot fungi [11]. Textile dyes-containing efuents to be treated with culture extracts or puried fungal enzymes often exhibit high pH due to the presence of auxiliar dyeing compounds while low pH is usual in fungal cultures as a result of their metabolism. However, I. resinosum culture liquid was able to decolorize not only pure solutions of synthetic dyes at acidic pH, but also synthetic dyebaths containing inorganic salts and the metal chelator EDTA. It can be concluded that I. resinosum as a producer of laccase and Mn-peroxidase is a promissing species for decolorization of textile dyes and dyebaths when used in combination with redox mediators. Acknowledgements This work was supported by the Czech-Slovenian S&T Cooperation Programme BI-SLO-CZ/05-06/022, the Ministry of Education, Youth and Sports of the Czech Republic (ME 873) and by the Institutional Research Concept AV0Z50200510 of the Institute of Microbiology, ASCR. References
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