Enzyme and Microbial Technology 39 (2006) 1023–1029

Production of ligninolytic enzymes by litter-decomposing fungiand their ability to decolorize synthetic dyes

Petr Baldrian ∗, Jaroslav SnajdrLaboratory of Biochemistry of Wood-Rotting Fungi, Institute of Microbiology ASCR, Vıdenska 1083, CZ-14220 Praha 4, Czech Republic

Received 31 October 2005; received in revised form 31 January 2006; accepted 7 February 2006

Abstract

Litter-decomposing basidiomycete fungi (LDF) including environmental isolates from oak forest soil were compared with white-rot fungi forligninolytic enzymes production and decolorization of synthetic dyes Poly B-411, Reactive Black 5, Reactive Orange 16 and Remazol BrilliantBlue R (RBBR). LDF differed significantly in laccase production. Mycena inclinata and Collybia dryophila produced significant amounts ofthe enzyme during the whole experiment, while the production in Stropharia rugosoannulata started after 3 weeks of cultivation. Soil isolatesexhibited detectable though very low laccase activity. The highest activity of Mn-peroxidase was detected in the cultures of C. dryophila with thepeak activities over 30 U l−1. In all other strains, Mn-peroxidase activity did not exceed 3 U l−1. The decolorization of 100 mg l−1 dyes after 28 daysroop©

K

1

tyees

wslrsasec

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anged 80–95% for RBBR, 60–95% for Poly B-411, 58–85% for Reactive Black 5 and 45–82% for Reactive Orange 16. The fastest degradationf Poly B-411 was performed by the strains with high levels of laccase and MnP while the decolorization of other dyes did not depend so strictlyn enzyme activities. The highest decolorization of azo dyes was achieved with the LDF C. dryophila, S. rugosoannulata and the soil isolates. Theresence of dyes significantly affected enzyme activities in fungal cultures.

2006 Elsevier Inc. All rights reserved.

eywords: Litter-decomposing fungi; Laccase; Mn-peroxidase; Decolorization; Dyes; Ecology

. Introduction

The possibility to use ligninolytic fungi for the decoloriza-ion of synthetic dyes attracted considerable attention in the pastears [1,2]. This was due to their production of ligninolyticnzymes – most frequently laccase and Mn-peroxidase – thatnable these microorganisms to oxidize a broad range of sub-trates including synthetic dyes [3,4].

Most studies so far focused on the possible use of the modelhite-rot species Phanerochaete chrysosporium, Trametes ver-

icolor, Pleurotus ostreatus and a few others. However, the usualaboratory studies do not reflect the fact that in order to achieveeal applicability, dye decolorization should occur under non-terile conditions of polluted water streams or soils. This isproblem, since white-rot fungi require a lignocellulose sub-

trate and many species of these typical wood colonizers do notxhibit satisfactory growth and competitivity under nonsterileonditions [5,6]. Even some strains of the best competitors P.

∗ Corresponding author. Tel.: +420 241062315; fax: +420 241062384.

ostreatus or T. versicolor failed to grow in the nonsterile envi-ronment [7,8]. The use of isolated enzymes does not solve theproblem since it is costly and the activity of isolated enzymescan rapidly decrease under nonsterile conditions [9].

The ecological group of litter-decomposing basidiomycetefungi (LDF) is represented by species closely physiologicallyrelated to white-rot fungi and inhabiting the natural environmentof soil and decaying litter [10]. A study of dye decolorization bythese fungi was not yet performed and LDF only rarely occurredin dye decolorization screenings [11,12]. The dye decoloriza-tion ability of these species can be anticipated from the reportsdescribing the presence of ligninolytic activities in these species[10,13] and from their ability to decolorize and degrade humicsubstances and PAH, compounds structurally related to dyes[10,14]. On the other hand, dye decolorization represents asimple test for fungal ability to transform lignin and humic sub-stances present in their natural environment.

The aim of this study was to describe the production of ligni-nolytic enzymes and decolorization of azo and anthraquinonedyes by selected species of LDF and to compare it with typ-ical white-rot species P. ostreatus and T. versicolor. The azo

E-mail address: baldrian@biomed.cas.cz (P. Baldrian). dyes Poly B-411 and Remazol Brilliant Blue R were used since

141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.02.011

1024 P. Baldrian, J. Snajdr / Enzyme and Microbial Technology 39 (2006) 1023–1029

their decolorization was frequently linked with the activity ofextracellular enzymes [15,16], Reactive Black 5 and ReactiveOrange 16 were used as the commonly used representatives ofreactive dyes recalcitrant to fungal degradation [17,18]. It shouldalso answer the question if the dye-decolorizing ability can befound among litter-decomposing basidiomycetes isolated fromthe natural environment (oak forest soil).

2. Materials and methods

2.1. Microorganisms

The strains of litter-decomposing fungi Collybia dryophila CCBAS811,Mycena inclinata CCBAS422 and Stropharia rugosoannulata CCBAS502, andwhite-rot fungi P. ostreatus CCBAS477 and T. versicolor CCBAS614 were fromthe Culture Collection of Basidiomycetes (Institute of Microbiology ASCR,Prague, Czech Republic).

Strains of ligninolytic fungi were isolated from forest litter. Litter layer(0–1 cm) from forest soil of oak (Quercus petraea) forest (Xaverov forest nat-ural reserve, Central Bohemia, Czech Republic) was collected. Three gramsof aliquots of mixed material were placed into Petri dishes and moistenedwith 5 ml sterile distilled water. Autoclaved (2 × 20 min, 121 ◦C) wood pieces,10 mm × 10 mm × 2 mm, soaked in 300 mg l−1 benomyl solution were placedon the top of the litter and Petri dishes were incubated until the wood pieceswere completely covered with mycelium [19]. Colonized wood pieces weresurface-sterilized by immersing in 5% H2O2 for 10 s to remove the surface-colonizing fungi. The pieces were then placed onto a MERBS agar plates(20 g l−1 malt extract, 0.5 g l−1 Rose Bengal, 3 mg l−1 streptomycine 15 g l−1

agar) and incubated at 25 ◦C until outgrowth from wood pieces was detectable.Tcmeb(tm

2

(M12B(t1Hflwgdrtm

2

tpfM

Fig. 1. Chemical structures of synthetic dyes.

(100 mM, pH 4.5) [22]. MBTH (3-methyl-2-benzothiazolinone hydrazone) andDMAB (3,3-dimethylaminobenzoic acid) were oxidatively coupled by the actionof the enzyme and formation of a purple indamine dye product was followedspectrophotometrically. The results were corrected by activities in test sampleswithout manganese where manganese sulfate was substituted by ethylenedi-aminetetraacetate to chelate Mn present in the samples. Lignin peroxidase (EC1.11.1.14) was assayed with veratryl alcohol as a substrate [23]. One unit ofenzyme activity was defined as the amount of enzyme catalyzing the formationof 1 �mol of the reaction product per min. Spectrophotometric measurementswere done in a microplate reader Sunrise (Tecan, Austria).

Visible spectra of culture liquid samples were recorded using a microplatereader Spectra Max Plus 384 (Molecular Devices, USA). The rate of decol-orization was expressed as the percentage decrease of absorbance maxima ofindividual dyes—592 nm for Remazol Brilliant Blue R, 597 nm for ReactiveBlack 5, 590 nm for Poly B-411 and 494 nm for Reactive Orange 16.

2.4. Decolorization of dyes by culture liquid and adsorption ofdyes to fungal mycelia

To study the contribution of extracellular enzymes to the decolorization ofsynthetic dyes, culture liquid from 28-days cultures of fungi was collected byfiltration and combined (1:1 v/v) with solutions of individual dyes (200 mg l−1).Incubation proceeded for 15 h at 25 ◦C in the dark. Absorbance at respectivemaxima was recorded for 15 h at 60 min intervals. The dye sorption by fun-gal mycelia during the decolorization process was determined using a bioticcontrol according to [24]. Briefly, fungal mycelium from 28-days cultures was

he isolates were microscopically checked for purity and the presence of clamponnections. Basidiomycete strains were inoculated onto ME agar (20 g l−1

alt extract, 15 g l−1 agar) containing 0.02% (w/v) ABTS (2,2′-azinobis-3-thylbenzothiazoline-6-sulfonic acid) to detect phenoloxidase activity and incu-ated at 25 ◦C in the dark. After 7 days, strains giving positive test with ABTSgreen color) were designated X1, X2, and X3, respectively, and used in fur-her studies. Both strains from the culture collection and natural isolates wereaintained on ME agar plates.

.2. Enzyme production and decolorization experiments

For preparation of inocula, the fungi were grown on HNHC agar plates20 g l−1 glucose, 2 g l−1 ammonium tartrate, 1 g l−1 KH2PO4, 0.5 g l−1

gSO4·7H2O, 0.2 g l−1 NaH2PO4, 50 mg l−1 CaCl2, 50 mg l−1, FeSO4·7H2O,0 mg l−1 CuSO4·5H2O, 5 mg l−1 ZnSO4·7H2O, 5 mg l−1 MnSO4·4H2O,5 g l−1 agar, pH 6.0) [20] at 25 ◦C for 7 days. The anthraquinone dyes Remazolrilliant Blue R (RBBR; Aldrich, dye content 45%, C.I. 61200) and Poly B-411

Sigma, dye content 65%) and azo dyes Reactive Black 5 (Aldrich, dye con-ent 55%, C.I. 20505) and Reactive Orange 16 (Aldrich, dye content 50%, C.I.7757) were used (Fig. 1). A 250-ml Erlenmeyer flask containing 40 ml of liquidNHC medium were supplemented with 100 mg l−1 of dye. Unsupplementedasks were used for enzyme activity measurements. Each flask was inoculatedith two mycelial agar plugs 7 mm in diameter (cut from the edge of an activelyrowing colony). The cultures were incubated without agitation at 25 ◦C in theark for 28 days. Each sampling day, 300 �l of the culture liquid was sterilelyemoved from each flask. At the end of cultivation, mycelia were separated fromhe culture liquid by filtration and dried at 105 ◦C until constant mass for dry

ass estimation. All treatments were run in triplicates.

.3. Enzyme activity and decolorization in whole fungal cultures

Laccase (EC 1.10.3.2) activity was measured by monitoring the oxida-ion of ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) in citrate-hosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0) [21]. Theormation of green radical was followed spectrophotometrically. Activity of

n-peroxidase (EC 1.11.1.13, MnP) was assayed in succinate-lactate buffer

P. Baldrian, J. Snajdr / Enzyme and Microbial Technology 39 (2006) 1023–1029 1025

Table 1Time course of laccase activities (U l−1) during the growth of litter-decomposing and white-rot fungi in liquid HNHC medium

Day 7 Day 10 Day 14 Day 17 Day 21 Day 24 Day 28

C. dryophila 48 ± 11 83 ± 11 40 ± 6 30 ± 1 56 ± 11 165 ± 23 140 ± 28M. inclinata 0.7 ± 0.1 1.5 ± 0.8 3.8 ± 1.2 5.4 ± 1.1 6.7 ± 1.4 5.2 ± 1.0 9.6 ± 1.2S. rugosoannulata 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.4 ± 2.1 8.1 ± 5.0 13.8 ± 5.3Strain X1 1.7 ± 0.2 2.7 ± 0.3 0.6 ± 0.6 0.5 ± 0.3 0.2 ± 0.2 0.3 ± 0.2 0.2 ± 0.2Strain X2 0.9 ± 0.1 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.0 0.2 ± 0.1Strain X3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.3 ± 0.0P. ostreatus 1.9 ± 0.9 9.7 ± 3.8 27.4 ± 9.3 38.5 ± 11.6 32.4 ± 12.5 21.0 ± 7.9 29.7 ± 7.0T. versicolor 60 ± 10 199 ± 31 280 ± 27 169 ± 12 423 ± 46 441 ± 30 522 ± 29

The data represent means and standard errors of means of three replicates.

collected and inactivated at 55 ◦C for 24 h. Dead mycelium was collected byfiltration, rinsed with distilled water and incubated with synthetic dyes solution(100 mg l−1) at 25 ◦C for 1 h. All experiments were run in triplicates.

3. Results

3.1. Production of ligninolytic enzymes in liquid cultures

Out of 10 isolates of litter-decomposing basidiomycete fungiwith the ability to colonize wood, only three strains exhibitedABTS oxidation as an indication of phenol oxidation. Thesestrains were used in later experiments in addition to the speciesfrom culture collections. All fungal species were able to growon liquid HNHC medium. The dry mass of mycelium after culti-vation was 0.90 g l−1 in C. dryophila, 1.18 g l−1 in M. inclinata,0.80 g l−1 in S. rugosoannulata, 1.00 g l−1 in P. ostreatus, and0.83 g l−1 in T. versicolor. Biomass production was remarkablyhigher in the soil isolates X1 and X2 (7.43 g l−1 and 7.50 g l−1,respectively), while it was only 0.38 g l−1 in X3.

In the cultures of white-rot fungi, laccase activities rangedin tens to hundreds of U l−1 (Table 1). Maximum activity in P.ostreatus was detected on day 17, while the activity increasedduring the whole course of cultivation in T. versicolor andreached over 500 U l−1. The litter-decomposing basidiomycetes(LDF) differed significantly in laccase production. M. incli-nata and C. dryophila produced significant amounts of theeSta

(

the cultures of T. versicolor and C. dryophila with the peak activ-ities over 30 U l−1. In all other strains, Mn-peroxidase activitydid not exceed 3 U l−1. No lignin peroxidase was detected infungal cultures.

3.2. Decolorization of synthetic dyes

With the exception of the strain X3, anthraquinone-baseddyes Poly B-411 and RBBR were decolorized to a greater extentthan azo dyes. The decolorization of RBBR ranged 80–98%after 28 days while it was 60–95% for Poly B-411, 58–85%for Reactive Black 5 and only 45–82% for Reactive Orange 16(Fig. 2). The fastest degradation of Poly B-411 was performedby the strains with stable and high levels of laccase and MnP—T.versicolor and the LDF C. dryophila. The decolorization by P.ostreatus, S. rugosoannulata and M. inclinata was slower butreached the same value in the end of the experiment. X1 andX2 cultures exhibited gradual color removal up to 60%, whilethe decolorization by X3 was negligible. The course of RBBRdecolorization was very similar, except that X1 and X2 culturesexhibited a very fast decolorization of the culture liquid.

The decolorization of azo dyes Reactive Black 5 and ReactiveOrange 16 did not depend so strictly on enzyme activities in indi-vidual cultures. T. versicolor was not among the best strains forReactive Black 5 and Reactive Orange 16 decolorization (Fig. 2).TtXa

d

TT ompo

CMSSSSPT

T

nzyme during the whole experiment, while the production in. rugosoannulata started after three weeks of cultivation. Allhree soil isolates exhibited detectable though very low laccasectivity (Table 1).

Mn-peroxidase was also produced by all tested fungiTable 2). The highest activity of Mn-peroxidase was detected in

able 2ime course of Mn-peroxidase activities (U l−1) during the growth of litter-dec

Day 7 Day 10 Day 14

. dryophila 6.4 ± 3.3 10.8 ± 3.3 5.3 ± 2.8. inclinata 0.2 ± 0.0 0.1 ± 0.0 0.4 ± 0.1

. rugosoannulata 1.9 ± 0.9 0.1 ± 0.1 0.3 ± 0.3train X1 0.1 ± 0.0 0.1 ± 0.1 0.1 ± 0.0train X2 0.6 ± 0.1 0.3 ± 0.1 0.0 ± 0.0train X3 0.3 ± 0.0 0.5 ± 0.1 0.5 ± 0.0. ostreatus 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0. versicolor 8.7 ± 4.7 20.9 ± 1.9 14.6 ± 5.7

he data represent means and standard errors of means of three replicates.

he highest decolorization of both azo dyes was achieved withhe LDF C. dryophila, S. rugosoannulata and the strains X1 and2. Interestingly, significant decolorization of both dyes was

lso achieved with the soil strain X3.While no significant effect of dyes on biomass dry mass was

etected, it strongly affected enzyme activities in fungal cultures.

sing and white-rot fungi in liquid HNHC medium

Day 17 Day 21 Day 24 Day 28

15.5 ± 7.3 20.0 ± 5.2 25.4 ± 3.7 33.0 ± 8.60.8 ± 0.1 0.7 ± 0.1 0.9 ± 0.1 1.2 ± 0.50.4 ± 0.4 0.6 ± 0.1 0.8 ± 0.6 2.5 ± 0.40.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.2 ± 0.20.0 ± 0.0 0.7 ± 0.1 0.7 ± 0.1 0.7 ± 0.10.5 ± 0.2 0.5 ± 0.0 0.7 ± 0.0 0.8 ± 0.00.6 ± 0.6 1.9 ± 0.6 1.6 ± 0.7 1.6 ± 0.1

33.2 ± 2.0 27.9 ± 10.1 29.9 ± 3.6 25.9 ± 8.8

1026 P. Baldrian, J. Snajdr / Enzyme and Microbial Technology 39 (2006) 1023–1029

Fig. 2. Decolorization of Poly B-411 (a), Reactive Black 5 (b), Remazol Brilliant Blue R (c) and Reactive Orange 16 (d) by white-rot and litter-decomposing fungi.C. dryophila (solid circles), M. inclinata (solid squares), S. rugosoannulata (open inverted triangles), strain X1 (open triangles), strain X2 (solid inverted triangles),strain X3 (open squares), P. ostreatus (solid triangles), T. versicolor (open circles). The data represent means of three replicates. Relative standard deviations wereless than 10%.

The effect of dyes was remarkably species-specific. Peak valuesof laccase activity in M. inclinata and S. rugosoannulata weresignificantly increased by all tested dyes while Poly-B411 alsoincreased laccase of X3 and P. ostreatus. The strains X1 andX2 on the other hand, showed significantly lower peak valuesin the presence of the same dye (Table 3). The presence of dyesalso increased the activity maximum of S. rugosoannulata Mn-peroxidase (more than 15-fold increase with Poly B-411). Alsothe strains X1 and X2 exhibited higher Mn-peroxidase maximain the presence of Poly B-411 and Reactive Black 5.

3.3. Contribution of extracellular enzymes and adsorptionto dye decolorization

The contribution of extracellular enzymes of fungi to dyedecolorization was determined as the ability of culture liquidto perform decolorization. The culture liquid samples from28-days cultivation of fungi exhibited significant differences

in dye decolorization performance (Table 4). In general, thefungi exhibiting high activities of laccase and Mn-peroxidasealso exhibited the highest decolorization with all tested dyes.Decolorization higher than 60% was found with the azo dyesPoly B-411 and RBBR incubated with culture liquid from bothwhite-rot fungi and the litter-decomposers C. dryophila and S.rugosoannulata. C. dryophila and T. versicolor also performedthe highest decolorization of Reactive Black 5 and ReactiveOrange 16, however, the decolorization was only 15–21% within15 h. Longer incubation did not substantially increase the decol-orization. Although the production of ligninolytic enzymes bysoil isolates X1–X3 was low, the fungi caused significant decol-orization of some dyes—the decolorization of Reactive Black5 and Reactive Orange 16 was comparable or higher than in P.ostreatus (Table 4).

The adsorption experiments confirmed that part of the decol-orization found in whole fungal cultures was due to the adsorp-tion of dyes to fungal mycelium. The strains X1 and X2 showed

P. Baldrian, J. Snajdr / Enzyme and Microbial Technology 39 (2006) 1023–1029 1027

Table 3Effect of synthetic dyes (100 mg l−1) on laccase and Mn-peroxidase activities (U l−1)

Control PB RB5 RBBR RO16

LaccaseC. dryophila 165 ± 23 336 ± 32 335 ± 18 525 ± 48 357 ± 52M. inclinata 10 ± 1 65 ± 10 46 ± 6 54 ± 8 48 ± 11S. rugosoannulata 14 ± 5 15 ± 3 87 ± 12 6 ± 2 19 ± 7Strain X1 2.7 ± 0.3 0.7 ± 0.2 0.2 ± 0.0 1.4 ± 0.3 1.7 ± 0.3Strain X2 0.9 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.6 ± 0.1 0.3 ± 0.0Strain X3 0.3 ± 0.0 1.3 ± 0.2 0.2 ± 0.0 0.3 ± 0.0 0.1 ± 0.0P. ostreatus 39 ± 12 78 ± 24 25 ± 3 128 ± 8 30 ± 6T. versicolor 522 ± 29 528 ± 41 577 ± 21 627 ± 17 557 ± 45

Mn-peroxidaseC. dryophila 33 ± 9 16 ± 2 8 ± 2 24 ± 1 62 ± 5M. inclinata 1.2 ± 0.5 0.1 ± 0.0 0.3 ± 0.1 4.7 ± 0.3 1.6 ± 0.2S. rugosoannulata 2.5 ± 0.4 6.6 ± 0.8 65.8 ± 7.1 9.9 ± 1.8 5.3 ± 1.1Strain X1 0.3 ± 0.0 3.0 ± 0.7 1.1 ± 0.2 0.3 ± 0.1 0.3 ± 0.1Strain X2 0.7 ± 0.1 3.0 ± 0.2 10.3 ± 3.0 1.0 ± 0.2 0.8 ± 0.0Strain X3 0.8 ± 0.0 0.8 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 1.1 ± 0.0P. ostreatus 1.9 ± 0.6 0.2 ± 0.1 0.8 ± 0.2 3.4 ± 0.5 6.3 ± 0.2T. versicolor 33 ± 2 36 ± 5 42 ± 1 41 ± 7 30 ± 5

Values represent the highest enzyme activities detected during 28-days cultivation in HNHC medium. The data represent means and standard errors of means of threereplicates. Abbreviations: PB, Poly B-411; RB5, Reactive Black 5; RBBR, Remazol Brilliant Blue R; RO16, Reactive Orange 16.

Table 4Activity of laccase and Mn-peroxidase (U l−1) and decolorization of dyes by the culture liquid of litter-decomposing and white-rot fungi cultivated for 28 days inliquid HNHC medium

Laccase (U l−1) MnP (U l−1) PB (%) RB5 (%) RBBR (%) RO16 (%)

C. Dryophila 128 ± 32 37 ± 11 81.3 ± 8.3 21.6 ± 0.4 81.6 ± 2.8 18.4 ± 0.6M. inclinata 6.3 ± 0.7 0.9 ± 0.2 42.4 ± 5.2 6.7 ± 1.2 20.6 ± 6.1 6.0 ± 0.5S. rugosoannulata 29 ± 2 2.2 ± 0.4 70.8 ± 1.2 11.8 ± 0.4 75.9 ± 2.8 11.6 ± 0.3Strain X1 0.3 ± 0.0 0.3 ± 0.0 17.0 ± 3.1 12.4 ± 2.4 7.9 ± 1.7 9.0 ± 1.5Strain X2 0.2 ± 0.1 0.3 ± 0.0 15.9 ± 0.9 11.0 ± 0.5 5.7 ± 0.8 5.9 ± 1.5Strain X3 0.2 ± 0.0 0.1 ± 0.1 4.7 ± 1.6 4.9 ± 0.7 9.5 ± 0.3 3.6 ± 0.4P. ostreatus 22 ± 6 1.6 ± 0.3 62.4 ± 16.8 9.7 ± 1.2 60.6 ± 5.1 6.1 ± .3.3T. versicolor 481 ± 37 21 ± 7 85.4 ± 3.0 20.8 ± 0.6 82.1 ± 1.9 15.2 ± 0.7

In decolorization assay, synthetic dyes (200 mg l−1) were incubated with culture liquid for 15 h at 25 ◦C. The data represent means and standard errors of means ofthree replicates. Abbreviations: PB, Poly B-411; RB5, Reactive Black 5; RBBR, Remazol Brilliant Blue R; RO16, Reactive Orange 16.

Table 5Mycelium dry mass and adsorption of synthetic dyes (100 mg l−1) to themycelium of litter-decomposing and white-rot fungi after 28 days of cultivation

Dry mass(g l−1)

PB (%) RB5(%)

RBBR(%)

RO16(%)

LaccaseC. dryophila 0.95 <2 10 17 6M. inclinata 0.78 <2 6 <2 <2S. rugosoannulata 0.97 <2 5 <2 <2Strain X1 7.50 33 79 79 60Strain X2 7.28 30 73 82 59Strain X3 0.39 <2 <2 <2 <2P. ostreatus 0.92 <2 13 <2 <2T. versicolor 1.01 19 10 25 3

Values represent means, relative standard deviations were less than 10%. Abbre-viations: PB, Poly B-411; RB5, Reactive Black 5; RBBR, Remazol Brilliant BlueR; RO16, Reactive Orange 16.

both high biomass production and high dye adsorption and thedecolorization found in their cultures is probably mainly due tomycelial adsorption. In other fungi the adsorption was usuallyless than 15% (Table 5).

4. Discussion

The wood-rotting white-rot fungi can be divided into sev-eral groups based on their production of the individual ligni-nolytic enzymes (lignin peroxidase, Mn-peroxidase and laccase)[25,26]. Litter-decomposing fungi differ from wood-rottingspecies with respect to their growth substrate, forest litter and soilthat is characterized by higher C:N ratio and microbial activity.Laccase is the most common ligninolytic enzyme among LDFand Mn-peroxidase is produced only by some species [4,27]. Inthis study, three strains out of ten wood-colonizing soil basid-iomycetes were able to oxidize ABTS, the seven others beingprobably cellulolytic or hemicellulolytic. The activities of lac-case and Mn-peroxidase was found in all tested LDF strains, butit differed by several orders of magnitude. Lignin peroxidase

1028 P. Baldrian, J. Snajdr / Enzyme and Microbial Technology 39 (2006) 1023–1029

was not detected (data not shown). The production under labo-ratory conditions, however, may not reflect the actual activitiesin the environment.

There are several reports about the oxidation of polycyclicaromatic hydrocarbons (PAH) and decolorization and degrada-tion of humic substances by LDF [10,13,14,27]. On the otherhand, qualitative data about decolorization of synthetic dyeswere so far reported only for a few species, e.g. Clitocybuladusenii and S. rugosoannulata [12]. The latter was found todecolorize efficiently the anthraquinonic dye Basic Blue 22 andthe azo dye Acid Red 183. S. rugosoannulata was also the bestdegrader of PAH among the species tested by Steffen et al. [14].All strains used in this study and their extracellular enzymeswere able to perform dye decolorization, although adsorption ofsome dyes to mycelium was responsible for most color removalby X1 and X2. Degradation of RBBR has been demonstratedto reflect laccase activity in T. versicolor cultures [16]. In ourstudy, the degradation of anthraquinonic dyes also reflected thequantity and time course of ligninolytic enzymes production.In this respect it has to be noted that the participation of Mn-peroxidase in xenobiotics degradation is also affected by theproduction of hydrogen peroxide by fungi [28] and the detectionof activity cannot be directly linked to degradation or decol-orization. Despite lower production of ligninolytic enzymes byLDF, C. dryophila and S. rugosoannulata were better azo dyedegraders than T. versicolor and P. ostreatus. It is clear thatlwldmbdw[tioplo

dlcstpnaofc

ops

is regarded as a reliable indicator of lignin degrading capability[34] and humic substances share structural properties with syn-thetic dyes. Further studies of dye decolorization by LDF couldthus help to understand their ecological role, find the best poten-tial degraders of synthetic dyes and confirm their applicabilityunder technologically relevant conditions.

Acknowledgements

This work was supported by the Czech Science Founda-tion (526/05/0168) and by the Institutional Research ConceptAV0Z50200510 of the Institute of Microbiology, ASCR.

References

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[2] Fu YZ, Viraraghavan T. Fungal decolorization of dye wastewaters: areview. Bioresour Technol 2001;79:251–62.

[3] Hofrichter M. Review: lignin conversion by manganese peroxidase(MnP). Enzyme Microb Technol 2002;30:454–66.

[4] Baldrian P. Fungal laccases occurence and properties. FEMS MicrobiolRev 2006;30:215–42.

[5] Lang E, Eller G, Zadrazil F. Lignocellulose decomposition and produc-tion of ligninolytic enzymes during interaction of white rot fungi withsoil microorganisms. Microb Ecol 1997;34:1–10.

[6] Martens R, Zadrazil F. Screening of white-rot fungi for their ability

[

[

[

[

[

[

[

[

[

itter-decomposing fungi represent a promissing alternative tohite-rot fungi with respect to dye decolorization. Although

igninolytic enzymes are assumed to be responsible for dyeecolorization, other mechanisms may also occur. The involve-ent of cytochrome P450 in decolorization of malachite green

y Cunninghamella elegans was recently demonstrated [29] andecolorization by reactive oxygen species produced by severalood-rotting basidiomycetes can possibly also play some role

30,31]. Enzyme activity in fungal cultures was affected byhe presence of dyes that caused in several cases a significantncrease of laccase and/or Mn-peroxidase activity. Such increasef activity can be also responsible for higher decolorization of aarticular dye. The induction can possibly be a result of physio-ogical changes due to dyes toxicity as earlier demonstrated forther xenobiotic compounds [32].

It was recently demonstrated that several products of ligninegradation could act as redox mediators and thus enhanceaccase-catalyzed decolorization [33]. The presence of theseompounds in soils is highly probable in addition to humic sub-tances derivatives of similar composition that can possibly playhe same role. This would enhance the decolorization of dyes inolluted soils by LDF producing predominantly laccase. In theonsterile soil environment, laccase production can also increases a result of interspecific interactions of basidiomycetes withther soil microbes. This phenomenon was demonstrated alsoor litter decomposers Agrocybe sp. and Mycena sp. and canontribute to dye decolorization [16].

The production of ligninolytic enzymes and decolorizationf dyes also indicates the ecological importance of LDF in therocesses of lignin breakdown and the metabolism of humicubstances. The decolorization of Poly B-411 used in this study

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