2003_d.wesenberg_white-rot fungi and their enzymes for the treatment of industrial dye effluents

8/10/2019 2003_D.wesenberg_White-rot Fungi and Their Enzymes for the Treatment of Industrial Dye Effluents

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White-rot fungi and their enzymes for the treatment of industrial dye effluents

Dirk Wesenberg, Irene Kyriakides, Spiros N. Agathos *

Bioengineering Unit (GEBI), Universite Catholique de Louvain, Place Croix du Sud 2/19, B-1348 Louvain-la-Neuve, Belgium

Abstract

White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. This ligninolytic system of white-rot fungi (WRF) is directlyinvolved in the degradation of various xenobiotic compounds and dyes. This review summarizes thestate of the art in the research and prospective use of WRF and their enzymes (lignin-modifyingenzymes, LME) for the treatment of industrial effluents, particularly dye containing effluents. The

textile industry, by far the most avid user of synthetic dyes, is in need of ecoefficient solutions for itscolored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks toemerging knowledge of the physiology of these organisms as well as of the biocatalysis and stabilitycharacteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.D 2003 Elsevier Inc. All rights reserved.

Keywords: Biodegradation; Dye decolorization; Mn peroxidases; Polyphenoloxidases (laccases); White-rot fungi;Textile effluent treatment; Detoxification; Immobilized cells; Bioreactors; Scale-up

1. Textile dyes and textile dye industry

In 1856 William Henry Perkin accidentally discovered the worlds first commerciallysuccessful synthetic dye. By the end of the 19th century, ten thousand new syntheticdyes had been developed and manufactured. Nowadays, India, the former USSR,

0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved.doi:10.1016/j.biotechadv.2003.08.011

* Corresponding author. Tel.: +32-10-47-3644; fax: +32-10-47-3062. E-mail address: [email protected] (S.N. Agathos).

www.elsevier.com/locate/biotechadvBiotechnology Advances 22 (2003) 161187


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industrial effluent discharge in combination with increasingly stringent legislation, makethe search for appropriate treatment technologies an important priority (ONeill et al.,1999) . Abiotic means of reduction of azo and other dyes exist but require highlyexpensive catalysts and reagents (Ro binson et al., 2001a) . A number of biotechnologicalapproaches have been suggested by recent research as of potential interest towards

combating this pollution source in an ecoefficient manner, including the use of bacteriaor fungi, often in combination with physicochemical processes (Willmott et al., 1998;McMullan et al., 2001; Robinson et al., 2001a; Borchert and Libra, 2001; Beydilli et al.,1998; Zissi and Lyberatos, 2001) . By far the single class of microorganisms most efficient in breaking down synthetic dyes are the white-rot fungi (WRF). Theseconstitute a diverse ecophysiological group comprising mostly basidiomycetous (and,to a lesser extent, litter-decomposing) fungi capable of extensive aerobic lignindepolymerization and mineralization. This property is based on the WRFs capacity to produce one or more extracellular lignin-modifying enzymes (LME), which, thanks to

Table 2Classes of organic dyes: structures of representative colorants

D. Wesenberg et al. / Biotechnology Advances 22 (2003) 161187 163


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their lack of substrate specificity, are also capable of degrading a wide range of xenobiotics.

2. WRF in ligninolysis and degradation of xenobiotics

2.1. Lignin-modifying enzymes

WRF are key regulators of the global C-cycle. Their LME, i.e., manganese peroxidases (MnP), E.C. 1.11.1.13; lignin peroxidases (LiP), E.C. 1.11.1.14 andlaccases (Lac), E.C. 1.10.3.2, are directly i nvolved not only in the degradation of lignin in their natural lignocellulosic substra tes (Becker and Sinitsyn, 1993; Hatak ka,1994) but also in the degradation of various xenobiotic compoun ds (Barr and Aust,

1994; Pointing, 2001; Scheibner et al., 1997) including dy es (Glenn and Gold, 19 83;Pasti-Grigsby et al., 1992; Paszczynski et al., 1992; Spadaro et al., 1992) . Some whiterot fungi produce all three LME while others produce only one or two of them(Hatakka, 19 94) . LME are essential for lignin degradation, however for ligninmineralization they often combine with other processes involving additional enzymes.Such auxiliary enzymes (by themselves unable to degrade lignin) are glyoxal oxidaseand superoxide dismutase for intracellular production of H 2O2, a cosubstrate of LiPand MnP, as well as glucose oxidase, aryl alcohol oxidase and cellobiose dehydro-genase involved in feedback circuits and linking ligninolysis with cellulose andhemicellulose degradation in natu re (Leonowicz et al., 19 99). In the interest of developing technological applications (e.g., delignification in the pulp and paper industry), lignin has been found to be partly mineralized in cell-free systems of LME, with considerably enhanced rates in the presence of cooxidants such as fattyacids (Kapich et al., 1999) or thiols (Hofrichter et al., 199 8a). In this way, an older concept of ligninolysis reemerges, enzymatic combustio n (Kirk and Farrell, 19 87).By extension, this enzyme-assisted process is applicable to the degradation of manyother recalcitrant molecules, such as synthetic dyes. Current views of LME are givenin two recent reviews with emphasis on L ac (Leonowicz et al., 20 01) or MnP(Hofrichter, 20 02).

The main LME are oxidoreductases, i.e., two types of peroxidases, LiP and MnP(Fig. 1), and a phenoloxidase, L ac (Fig. 2). The physiology of LME production byWRF for ligninolysis or recalcitrant pollutant degradation has been studied extensively.In summary, a number of more-or-less general statements can be made despite the

many exceptions that are due to the wide variety of fungal taxa and of experimentalconditions reported: LME are produced by WRF during their secondary metabolismsince lignin oxidation provides no net energy to the fungus; synthesis and secretion of these enzymes is often induced by limited nutrient levels (mostly C or N); productionof LiP and MnP is generally optimal at high oxygen tension but is repressed byagitation in submerged WRF liquid culture, while Lac production is often enhanced byagitation; frequently, more than one isoforms of LME are expressed by different taxaand culture conditions. These features are important in the process design andoptimization of fungal treatment of colorant-containing effluents.



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The most common ligninolytic peroxidases produced by almost all white-rot basidio-mycetes and by various litter-decomposi ng fungi are man ganese peroxidases (MnP).These are glycosylated glyc oproteins (Nie et al., 1999) wit h an iron protoporphyrin IX(heme) p rosthetic group (Glenn and Gold, 1985) , molecular wei ghts between 32 and 62.5kDa (Hofrichter, 2002) and are secreted in multiple isoforms (Leisola et al., 1987; Ur zua et al., 1995) . MnP preferentially oxidize Mn 2 + into Mn 3 + (Glenn e t al., 1986) , which isstabilized by chelators such as oxalic acid (Wariishi et al., 1992) , itself also excreted by thefungi (Galkin et al., 1998; Kuan and Tien, 1993; Takao, 1965) . Chelated Mn 3 + acts as a

highly reactive (up to 1510 mV in H 2O, Cui and Dolphin, 1990 ) low molecular weight,diffusible redox-mediator. Thus, MnP are able to oxidize and depolymerize their natural

Fig. 1. Generic scheme of the catalytic cycle of peroxidases.

Fig. 2. The catalytic cycle of laccases.



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substrate, i.e., lignin as well as recalcitrant xenobiotics such a s nitroaminotolue nes(Scheibner et al., 1997; Van Aken et al., 19 99) and textile dyes (Heinfling et al.,

1998a) . In vitro depolymerization can be enhanced in the presen ce of cooxidants s uchas thiols (e.g., glutathione) or unsaturated fatty acids (e.g., Tween 80) (Hofrichter, 20 02).

Lignin peroxidases (LiP) catalyze the oxidation of nonphenolic aromatic ligninmoieties and similar compounds. LiP are well known as part of the ligninolytic syst em both of aphyllophoralic and agaricalic fu ngi (Glenn et al., 1983; Hatakka et al., 19 87;Hofrichter and Fritsche, 1997) . The extracellular N-glycosylated LiP with molecular masses between 38 and 47 kDa cont ain heme in the active site and show a classical peroxidase mechanis m (Tien et al., 1 986, Fig. 1). LiP catalyze seve ral oxidations in theside chains of lignin and relate d compounds (Tien and Kirk, 1983) by one-electronabstraction to form reactive radi cals (Kersten et al., 1985) . Also the cleavage of aromaticring structures has been report ed (Umezawa and Higuchi, 19 87). The role of LiP in

ligninolysis could be the further transformation of lignin fragments which are initiallyreleased by MnP. LiP are not essential for the attack on lignin: several highly active WRFand litter-decaying fungi (e.g., Ceriopsis subvermispora , Dichotomitus squalens , Panustigrinus , Rigidosporus lignosus ) do not excrete this enzy me (Galliano et al., 19 91;Hatakka, 1994; Maltseva et al., 1991; Pe rie and Gold, 1991) . LiP have been used tomineralize a variety of recalcitrant aromatic compounds, such as three- and four-ring PAHs(Gunther et al., 19 98), polychlorinated biphen yls (Krcma r and Ulrich, 19 98) and dyes(Chivukula et al., 19 95). 2-Chloro-1,4-dimethoxybenzene, a natural metabolite of WRF isreported to act as a redox mediator in the LiP-catalyzed oxidati ons (Teunissen et al., 19 98).A third group of peroxidases, versatile peroxidases (VP), has been recently recognized,that can be regarded as hybrid between MnP and LiP, since they can oxidize not onlyMn 2 + but also phenolic and nonphenolic aromatic compounds including dyes. VP have been described in species of Pleurotus and Bjerkand era (Heinfling et al., 1998a,b; Me ster and Field, 1998) . A comprehensive review of the molecular biology of WRF peroxidasesis given by Martnez (2002) and by Conesa et al. (20 02).

Fungal laccases as part of the ligninolytic enzyme system are produced by almost allwood- and litter-transforming basidiomycetes. This group of N-glycosylated extracellular blue oxidases with molecular masses of 60390 k Da (Call and Mu cke, 1997; Reinh am-mar, 1984) , contain four copper atoms in the active site (as Cu 2 + in the resting enzyme)that are distributed among different binding sites, and are classified into three types withdifferential specific characteristic propert ies (McGuirl and Dooley, 1999; Messerschm idt,1997) . Laccases catalyze the oxidation of a variety of aromatic hydrogen donors with theconcomitant reduction of oxygen to wa ter (Fig. 2). Moreover, laccases do not only oxidize

phenolic and methoxyphenolic acids, but also decarboxylate them and attack their methoxy groups (demethylation). Laccases have been intensively studied with a focuson their industrial applicabil ity (Bajpai, 1999; Gianfreda et al., 1999; Rodr guez et al.,1999; Yaropolov et al., 1994) , molecular genet ics (Cullen, 1997; Karahanian et al., 19 98;Ong et al., 1997; Collins and Dobson, 1997) and cloning (Hatamoto et al., 19 99). Laccaseshave been reported to oxidize many recalcitrant substances, such as chlorophen ols (Fahr et al., 1999; Grey et al., 1998; Ricotta et al., 1996; Roy-Arcand and Archibald, 1991) , PAHs(Majcherczyk et al., 19 98), lignin-related structu res (Bourbonnais et al., 1996; Boyle et al.,1992) , organophosphorous compoun ds (Amitai et al., 19 98), nonphenolic lignin model



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compounds (Kawai et al., 1988; Majcherczyk et al., 1 999) , phenols (Bollag et al., 1988;Xu, 1996) and last but not least, aromatic dyes (Abadulla et al., 2000; Chivukula and

Renganathan, 1995; Rodr guez et al., 1999) . A comparative summary of the maincharacteristics of LME from WRF is given in Tabl e 3.

2.2. Small-molecule mediators

Given the random polymer nature of lignin and the bulk of LME, direct and specificinteractions between lignin (or recalcitrant structural analogs) and LME are highly

Table 3Comparison of the properties of MnP, LiP and Lac from WRF

E.C. MnP 1.11.1.13 LiP 1.11.1.14 Lac 1.10.3.2Mn(II): H 2O2oxidoreductases

diarylpropan O 2, H2O2oxidoreductases

p-benzendiol: O 2-oxidoreductases

Prosthetic group Heme Heme 1 type-1-Cu, 1 type-2-Cu,2 coupled type-3-Cu,

MW (kDa) 32 a 62.5 b (122 a ) 38 47 59 110 (tetramers V 390 c

Glycosylation N N N Isoforms monomers;

up to 11dmonomers; up to 15 mono-, di-, tetramers; several

p I 2.8e 7.2f 3.2 4.7 2.6 4.5 pH range 2.6g 4.5h 2.0 5.0 2.0 8.5E0 (mV) 1510 i 1450 j 500800 k

C C cleavage yes yes no

H2O2-regulated yes yes noStability + + + + + + + Native mediators Mn 2 + ; Mn3 + VA? l, 2Cl-14DMB m 3-HAA n

Specificity Mn 2 + broad, aromatics,incl. nonphenolics

broad, phenolics

Secondary andsynthetic mediators

Thiols, unsaturatedfatty acids

NO ABTS o, HBTo, syringaldazine

(Modified from Fakoussa and Hofrichter, 1999 ).a Basidiomycete strain RBS k1 (Willmann and Fakoussa, 1997) . b Ceriporiopsis subvermispora in SSF (Lobos et al., 1994) .c (Thurston, 1994) .d Ceriporiopsis subvermispora (Urzua et al., 1995).e Nematoloma frowardii (Schneega et al., 1997) .f Panaeolus sphinctrinus (Heinzkill et al., 1998) .g P. tigrinus (Maltseva et al., 1991) .h Pleurotus ostreatus (Sarkar et al., 1997).i Chelator H 2O (Cui and Dolphin, 1990) . j (Schoemaker and Leisola, 1990) . VA: Veratryl alcohol.k (Messerschmidt, 1997) .l (Farrell et al., 1989; Tien and Kirk, 1983) .m (Teunissen and Field, 1998; Teunissen et al., 1998b) . 2Cl-14DMB:2-chloro-1,4-dimethoxybenzene.n (Eggert et al., 1995) . 3-HAA:3-hydroxyanthranilic acid.o ABTS: 2,2 V-azinobis(3-ethylbenzthiazoline-6-sulfonate); HBT:1-hydroxybenzotriazole (Bourbonnais et al.,

1996) .



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improba ble (Evans and Hedger, 20 01). Rather low-molecular weight, diffusible redoxmediators provide high redox potentials (>900 mV) to attack lignin and are able to

migrate into the l ignocel lulose complex. Examples of native as well as syntheticmediators are given in Table 4. They could be involved in the LME-catalyzed generationof reactive radical moieties from a variety of lignin-like substrates, but also in theformation of reactive oxygen spec ies (ROS) which either directly or indirectly co uldattack lignin or xenobiotic molecu les (Hammel, 1996; Van Aken and Agathos, 20 01,2002) .

Organic acids, excreted by several fungal organisms, chelate and stabilize Mn 3 + . MnPwas found to simultaneously decompose organic acids (such as malonate) oxidatively andoxidize Mn 2 + to Mn3 + even in the absence of H 2O2. Thus, organic acids are postulated to be the origin of carbon-centered radicals (acetic acid radicals, COOH C

S

H2, Reaction 1), peroxyl radicals (COOH CH 2OO

S , Reaction 2), superoxide (O 2S

, Reactions 5 and 8),

formate radicals (CO 2S

, Reactions 6 and 7). Such radicals could be a source of peroxides,which can be used by MnP as substrates instead of H 2O2. Consequently, even fungi

Table 4 Native and synthetic mediators in LME systems

Mediator Organism (enzyme) Reference

Native mediatorsMn3 + Phanerochaete

chrysosporium (MnP)Wariishi et al., 1992

Organic acids(malonate, oxalate, etc.)

Armillaria mellea, Fomes annosus, Pleurotus ostreatus,

Phanerochaete chrysosporium, Phlebia radiata, Cenporiopsis subvermispora, Nematoloma frowardii (LiP, MnP)

Galkin et al., 1998;Hofrichter et al., 1999;

Takao, 1965

Veratryl alcohol Phanerochaete chrysosporium (LiP) Lundquist and Kirk, 19 783-Hydroxyanthranilic acid

(3-HAA) Pycnoporus cinnabarinus (Lac) Eggert et al., 1996;

Eggert et al., 19972-Chloro-1,4-

dimethoxybenzene(2Cl-14DMB)

Trametes versicolor (LiP) Teunissen and Field, 1 998

Synthetic mediators1-Hydroxybenzotriazole

(1-HBT)Trametes versicolor, Trametesvillosa, Pycnoporus cinnabarinus,

Botrytis cinerea, Myceliophthorathermophila, Coriolopsis gallica, Pleurotus ostreatus variousorganisms (Lac)

Bourbonnais et al., 199 6;Crestini and Argyropoulos,

1998; Li et al., 1999;Pickard et al., 1999

Violuric acid Trametes villosa, Pycnoporuscinnabarinus, Botrytis cinerea, Myceliophthora thermophila (Lac)

Li et al., 1999

2,2 V-Azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS)

Trametes versicolor, Coriolopsis gallica, Pleurotus ostreatus,various organisms (Lac)

Bourbonnais et al., 199 6;Crestini and Argyropoulos,1998; Pickard et al., 1999



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obv iously lacking H 2O2 -generating oxidases could be efficient lignin-degraders(Hofrichter et al., 1998b) and , by extension, useful in the degradation of xenobiotics such

as dyes.

COOH CH2 COOH Mn 3 ! COOH CS

H2 CO 2 H Mn 2

Reaction 1

COOH CS

H2 O2 ! COOH CH 2OOS

Reaction 2

COOH CH 2OO Mn 2 ! COOH CH2OOH Mn 3 Reaction 3

COOH CH 2OOH 2 Mn 2 ! COOH CHO H2O 2 Mn 3 Reaction 4

COOH CH 2OOS

O2 ! COOH COOH OS

2 H Reaction 5

COOH CHO Mn3 1=2 O2 ! HCOOH COS

2 Mn2 Reaction 6

COOH COOH Mn3

! CO2 COS

2 Mn2

Reaction 7

COS

2 O2 ! CO2 OS

2 Reaction 8

OS

2 Mn2 2 H ! H2O2 Mn 3 Reaction 9

H2O2 2 Mn 2 ! H2O 2 Mn 3 Reaction 10

On the other hand organic acids (e.g., oxalate) chelate cations including Fe2 +

(Dut ton et al., 1993) , therefore such acids are indirectly involved in the regulation of Fentonsreaction due to regulation of Fe 2 + concentration (Fenton, 1894; Koenigs, 1972) , whichsupplies fungal degradation reactions with hydronium ions (H 3O

+) and hydroxyl radicals(HO

S, HO ). Recent evidence strongly suggests the involvement of formyl and superoxide

free radicals in the in vitro mineralization of recalcitrant nitroaminoaromatic molecules byMnP or by its biomimetic analog Mn(III)/oxalate/O 2 (Van Aken and Agathos, 2002) .

The in vitro degradation of lignin and other recalcitrant molecules by MnPs isconsiderably enhanced in the presence of thiols [reduced glutathione (GSH), cysteine



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Table 5Selected WRF, able to decolorize synthetic (textile) dyes

Organism LMEa

(Reference) Dye b

(Reference) Bjerk andera adusta |LiP|MnP

(Kaal et al., 1995)Reactive Orange 96 N = N , Reactive Violet 5 N = N ,Reactive Black 5 N = N , Reactive Blue 15 PC , ReactiveBlue 38 PC (Heinfling et al., 1997)Remazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotnyet al., 2001)Reactive Violet 5 N = N , Reactive Black 5 N = N , ReactiveBlue 38 PC by MnP (Heinfling et al., 1998a,b)

Bjerk andera sp. | |MnP(Moreira et al., 2000)

Orange II N = N , Reactive Blue 38 PC , Poly R-478 PAQ

(Moreira et al., 2000)Ceri poria

metamorphosaRemazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotnyet al., 2001)

Daed alea flavida ND Brilliant Green TPM , Cresol Red TPM , Congo Red N = N

(Gill et al., 2002) Daed aleopsis

confragosaRemazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotnyet al., 2001)

Dichomitus squalens Lac| |MnP (Perieand Gold, 1991)

Brilliant Green TPM , Cresol Red TPM , Crystal Violet TPM ,Congo Red N = N , Orange II N = N (Gill et al., 2002)

Geot richumcandidum

(Kim and Kim, 1995 ) Reactive Blue 5 PAQ (Kim and Shoda, 1999)

Irpex lacteus Lac|LiP|MnP(Novotny et al., 2000)

Methyl red N = N , Congo Red N = N , Naphtol BlueBlack N = N , Remazol Brilliant Blue R PAQ , Bromophenol blueTPM , Copper (II) phthalocyaninetetrasulfonic acidtetrasodium salt MC , Poly R-478 PAQ (Novotny et al., 2001)

Lentinus tigrinus Lac| |MnP (Moreiraet al., 2000)


(Moreira et al., 2000) Mycoacia nothofagi Remazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotny

et al., 2001) Phan erochaete |LiP|MnP (Kaal Remazol Turquoise Blue PC (Conneely et al., 1999)

chrysosporium et al., 1995)(Lac)|LiP|MnP(Cameron et al., 2000)

azo dyes, Azure Blue, Cresol Red TPM , CrystalViolet TPM , Bromophenol blue TPM (Cameron et al., 2000)Acid Green 27 PAQ , Copper phtalocyanine tetrasulphonicacid tetrasodium salt MC , Indigo Carmine, NeutralRedHC , Acid Red 106 N = N , Mordant Yellow 10 N = N ,Brilliant Yellow N = N , Chrysophenine N = N , ChlorazolYellow N = N , Cibacron Brilliant Yellow 3G-P (ReactiveYellow 2) N = N;HC , Cibacron Brilliant Red 3BA(Reactive Red 4) N = N;HC , Orange II N = N , CrystalViolet TPM , Brilliant Green TPM (Knapp et al., 1995)Remazol Brilliant Blue R PAQ (Novotny et al., 2001)Indigo (Balan and Monteiro, 2001)

Lac| |MnP (Chagasand Durrant, 2001)

Amaranth N = N , New Coccine N = N , Orange G N = N ,Tartrazine N = N (Chagas and Durrant, 2001)Reactofix Orange N = N , Reactofix Golden Yellow N = N ,Reactofix Blue HE 2R

N = N , Navilene Black N = N , Sulphur Green N = N , Sulphur Red N = N , Navione Blue N = N , Vat Brown N = N (Capalash and Sharma, 1992)Red 2B AB (Jain et al., 2000)Congo Red N = N (Gill et al., 2002)

(continued on next page)



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Organism LME a (Reference) Dye b (Reference)

Reactive Orange 96 N = N , Reactive Violet 5 N = N ,Reactive Black 5 N = N , Reactive Blue 15 PC , ReactiveBlue 38PC (Heinfling et al., 1997)Everzol Turquoise Blue G PC , Everzol Yellow 4GL,Everzol Red RBN, Orange K-GL, Everdirect SupraYellow PG (Kapdan et al., 2000a)Orange II N = N , Reactive Blue 38 PC , Poly R-478 PAQ

(Moreira et al., 2000) Phanerochaete

sordida| |MnP (Moreira

et al., 2000)Orange II N = N , Reactive Blue 38 PC , Poly R-478 PAQ

(Moreira et al., 2000) Phellinus gilvus Indigo (Balan and Monteiro, 2001) Phellinus

pseudopunctatusRemazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novo tnyet al., 2001)

Phlebia brevispora Brilliant Green TPM , Cresol Red TPM , Crystal Violet TPM

(Gill et al., 2002) Phlebia (Merulius)

tremellosaLac|LiP|MnP(Ralph et al., 1996;Vares et al., 1994)

Cibacron Red, Remazol Navy Blue, Remazol Red,Cibacron Orange, Remazol Golden Yellow, Remaz olBlue, Remazol Turquoise Blue, Remazol Black B,Mixture (Kirby et al., 2000)

Phlebia fascicularia Brilliant Green TPM , Cresol Red TPM , Crystal Violet TPM ,Congo Red N = N , Orange II N = N (Gill et al., 2002)

Phlebia floridensis Brilliant Green TPM , Cresol Red TPM , Crystal Violet TPM ,Congo Red N = N , Orange II N = N (Gill et al., 2002)

Phlebia radiata Lac| |MnP (Moreiraet al., 2000)


(Moreira et al., 2000)Crystal Violet TPM , Congo Red N = N , Orange II N = N (Gillet al., 2002)

Piptoporus betulinus Acid Green 27PAQ

, Copper phtalocyanine tetrasulphonicacid tetrasodium salt MC , Indigo Carmine, Acid Red106 N = N , Brilliant Yellow N = N , Chrysophenine N = N ,Chlorazol Yellow N = N , Cibacron Brilliant Yellow 3G-P(Reactive Yellow 2) N = N;HC , Cibacron Brilliant Red 3B A (Reactive Red 4) N = N;HC , Orange II N = N , Cryst alViolet TPM , Brilliant Green TPM (Knapp et al., 1995)

Pleurotus eryngii |LiP|MnP (Mart-nez et al., 1996; Hein-fling et al., 1998a,b)

Reactive Violet 5 N = N , Reactive Black 5 N = N , React iveBlue 38PC by MnP (Heinfling et al., 1998a,b)

Pleurotus ostreatus | |MnP? (Ha et al., 2001; Sarkar et al., 1997)

Remazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novo tnyet al., 2001)Acid Green 27 PAQ , Copper phtalocyanine tetrasulphonicacid tetrasodium salt MC , Indigo Carmine, Neutral

RedHC , Mordant Yellow 10 N = N , Brilliant Yellow N = N ,Chrysophenine N = N , Cibacron Brilliant Yellow 3G-P(Reactive Yellow 2) N = N; HC , Cibacron Brilliant Red 3B A (Reactive Red 4) N = N;HC , Orange II N = N , Cryst alViolet TPM , Brilliant Green TPM (Knapp et al., 1995)

Pleurotus sajor-caju Lac| | (Chagas andDurrant, 2001)

Amaranth N = N , New Coccine N = N , Orange G N = N ,Tartrazine N = N (Chagas and Durrant, 2001)

| |MnP? (Boyleet al., 1992)

Indigo (Balan and Monteiro, 2001)

Table 5 (continued )



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systems from WRF culture supernatants. LME-producing profiles vary. For instance, Lacwas the main enzyme involved in dye decolorization by cultures of Phlebia tremellosa

(Kir by et al., 2000; Robinson et al., 2001b) and by Pleurotus sajorcaju (Chagas andDurrant, 2001) , whereas LiP or MnP activity was absent (Kir by et al., 2000) . MnP couldonly be detected when the culture medium was supplemented with MnCl 2. Elsewhere, the presence of LiP and/or MnP in addition to Lac ( Pleurotus ostreatus , Schizophyllumcommune , Sclerotium rolfsii , Neurospora crassa ) seemed to increase by up to 25% thedegree of decolorization of individual commercial triarylmethane, anthraquinonic, andindigoid textile dyes using enzyme preparations (Abadulla et al., 2000) . On the contrary,MnP was reported as the main enzyme involved in dye decolorization by Phanerochaetechrysosporium (Chagas and Durrant, 2001) and LiP for Bjerkandera adusta (Ro binson et

Organism LME a (Reference) Dye b (Reference)

Poly porus ciliatus Lac| |MnP (Moreiraet al., 2000)

Orange II N = N , Reactive Blue 38 PC , Poly R-478 PAQ(Moreira et al., 2000)

Poly porus sanguineus

Brilliant Green TPM , Crystal Violet TPM , Congo Red N = N

Brilliant Green TPM , Crystal Violet TPM , Congo Red N = N

(Gill et al., 2002) Pycnoporus

sanguineusOrange G N = N , Amaranth N = N , Bromophenol Blue TPM

Malachite Green TPM (Pointing and Vrijmoed, 2000)Indigo (Balan and Monteiro, 2001)

Stere um hirsutum Lac| |MnP (Moreiraet al., 2000)


(Moreira et al., 2000)Stere um rugosum Remazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotny

et al., 2001)Tram etes (Coriolus )

versicolor

Lac|LiP|MnP

(Hatakka, 1994)

Everzol Turquoise Blue G PC , Everzol Yellow 4GL,

Everzol Red RBN, Orange K-GL, Everdirect SupraYellow PG (Kapdan et al., 2000a,b)Acid Green 27 PAQ , Copper phtalocyanine tetrasulphonicacid tetrasodium salt MC , Indigo Carmine, NeutralRedHC , Acid Red 106 N = N , Mordant Yellow 10 N = N ,Brilliant Yellow N = N , Chrysophenine N = N , ChlorazolYellow N = N , Cibacron Brilliant Yellow 3G-P (ReactiveYellow 2) N = N; HC , Cibacron Brilliant Red 3B A(Reactive Red 4) N = N; HC , Orange II N = N , CrystalViolet TPM , Brilliant Green TPM (Knapp et al., 1995)Amaranth N = N , Remazol Black N = N , Remazol Brilliant Blue PAQ , Reactive Blue 15 PC , Tropaeolin N = N , RemazolOrange N = N , Remazol Brilliant Red BB N = N (Swamyand Ramsay, 1999)

Reactive Orange 96 N = N

, Reactive Violet 5 N = N

,Reactive Black 5 N = N , Reactive Blue 15 PC , ReactiveBlue 38 PC (Heinfling et al., 1997)Remazol Brilliant Blue R PAQ , Poly R-478 PAQ (Novotnyet al., 2001)

ND not determined.a Appearance of LME: Lac|LiP|MnP: all LME; | | : no LME. b Dyes, grouped into N = N : (di)azo dye, PC : phthalocyanine dye, MC metal complex dye, PAQ :

(poly)anthraquinone dye, TPM : triphenylmethane dye, HC : heterocyclic dye, AB) : acrylic basic dye.

Table 5 (continued )



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al., 2001b) . LiP was also conside red as the principal decolorizing enzyme in cultures of P.chrysospori um (K irby et al., 1995) . It is clear that LME play significant roles in dye

metabolism by W RF (McMullan et al., 20 01). This is not surprising, given the structuralsimilarity of most commercially important dye s (Tabl e 2) to lignin (sub)structuresamenable to transformation by LME, as described in the preceding sections. In vitrodecolorizations using LME were examined, e.g., using Lac from Pyricularia oryzae(Chivukula and Renganathan, 19 95) and Trametes versicolor (Wesenberg et al., in preparation), LiP from P. chrysospori um (Chivukula et al., 1995; Heikkila et al., 19 98)and MnP (or, more accurately, VP) from B. adusta and Pleurotus eryn gii (Heinfling et al.,1998a,b) . Of most interest for practical applications a ppears to be the different e nzymatic pattern depending on the lign inolytic strains us ed (McMullan et al., 20 01) . Dyemineralization by WR F was confirmed by 14 C-ring-labeled azo dyes that were mineralizedusing P. chrysospori um (Spadaro et al., 19 92). The influence or not of the substitution

pattern on the dye mineralization rates is a matter of controve rsy (Paszczynski et al., 19 92;Spadaro et al., 1992) , though it is clear that dye decolorization is not equivalent to dyemineralization. There is a definite gap in our current knowledge of decolorization and,even more so, of mineralization mechanisms. With a lack of insight concerning potentiallytoxic albeit colorless accumulating intermediates, our capacity to evaluate the truetechnical potential of WRF and their LME remains incomplete.

However, these difficulties are even greater if one considers that complex mixedeffluents are extremely variable in composition in one and the same factory, as is oftenthe case in the textile industry. Thus, the decolorization of real effluents requires anappropriate choice of fungal strain as well as of reactor environment. Real textile dyeeffluents contain not only dyes but also salts, sometimes at very high ionic strength andextreme pH values, chelating agents, precursors, by-products, surfactants, etc. Asreported in a systematic study by Knapp et al. (19 95), certain WRF strongly decolorize particular dyes but not others, whereas certain strains are more comprehensive in their decolorizing capacities. Small structural differences in dye mixtures can markedly affect decolorization, and this may be due to electron distribution and charge density,although steric factors may also contribute. In another report, Cu and Fe chelators aswell as anionic detergents, which could be found in real textile industrial effluents,inhibited Polyporus sp. and Trametes villosa up to 20%, whereas S. commune LMEswere inhibited up to 70 % (Abadulla et al., 20 00) . Thus, in spite of the highdecolorization efficiency of some strains, decolorizing a real industrial effluent is quitetroubleso me. Knapp and Newby (19 99) were the first to report the decolorization of aneffluent of the chemical industry containing an azo-chromophore by WRF. Only

recently the first attempt to apply WRF to decolorize a real textile dye industrialeffluent was published by our group. Using the agaric white-rot fungus, Clitocybuladusenii maximal decolorization rates were achieved over a period of 20 days at 28 j Cusing fourfold diluted dye-containing effluent (6559 color units as defined in StandardMetho ds (Clesceri et al., 19 98)) on a 5-day pregrown myceliu m (Wesenberg et al.,2002) (Fig . 3). The main enzyme involved in the decolorization achieved by C. duseniiwas considered to be Lac. A typical Lac production pattern, seen in T. versicolor but exhibited by all efficient dye-decolorizer WRF, is given in Fig. 4. However, MnPappeared to be induced at higher effluent concentrations and might also have a role in



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the decolorization by C. dusenii (Wesenberg et al., 2002) . The extraction of theseenzymes allowed a first investigation of biochemical features of the Lac of several newdye effluent-decolorizing WRF (Tab le 6).

The inherent complexity of both the dyes structures and the enzymatic transformationmechanisms makes the elucidation of the degradation pathways a difficult task. Possiblenon-LME degrading mechanisms have been suggested (Pasti and Crawford, 1991) and dyedegradation-specific enzymes have been described, e.g., Remazol Brilliant Blue R (RBBR) decolorizing peroxidase from P. ostreatus (Kwang-Soo and Chang-Jin, 1998;

Vyas and Molitoris, 1995) . Several workers have attempted to correlate the production of LME in WRF and the rates of decolorization. In a recent work by Lorenzo et al. (2002) it was shown that it was possible to stimulate the yield of Lac activity of T. versicolor by

Fig. 3. Decolorization of raw effluent by C. dusenii . Decolorization of raw wastewater in modified Kirk medium by C. dusenii after incubation times of 10 and 20 days based on color units (filled symbols, left-hand axis) basedon a reference to K 2PtCl 6 as well as on the measured absorbance as derived from the integrated surface area of

spectral scans (400700 nm; empty symbols, right-hand axis). Th e cultures contained 10% (.

/ o

), 25% (E

/ 4 )or 33% (n / 5 ) of raw wastewater in modified Kirk medium (Wesenberg et al., 2002) .

Fig. 4. Typical profile of laccase production by WRF. Laccase activity during culture of Trametes versicolor witha draw-fill method. When the laccase activity ( . . ) reached its maximum, the culture liquid was harvested andreplaced by fresh medium (- - - - -).



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using several agricultural wastes, however the decolorizing capacity of the extracellular liquid did not appear to be proportionately increased. A direct correlation between LME production and industrial effluent decolorization was given by Wesenberg et al. (20 02),suggesting a differential inducing effect of the effluent on the LME production pattern(Fig. 5). An earlier study of Wong and Yu (19 99) proposed a mechanism for theincreased decolorization capacity of T. versicolor Lac, that involves the decolorizationof nonsubstrate dyes in effluents via substrate dyes which also act as mediators in theLac catalytic cycle. Further investigation is needed to define the structures of dyes,which could be Lac mediators, so that the efficiency of mixed dye effluents could be predicted.

WRF are superior dye-decolorizers in comparison with prokaryotes. Even the lignin-transforming actinomycete Streptomyces chromofuscus is a weak decolorizer comparedto P. chrysospori um (Paszczynski et al., 19 92), whose decolorizing capacity is due toLiP and not M nP (Young and Jian, 19 97). Immunochemical methods have revealed that a fraction of the LiP produced by P. chrysosporium remains associated with the fungalwall (Garcia et al., 19 87) and washed pellets have been shown to retain partial lignin-degrading abil ity (Kurek and Odier, 19 90). Although several works refer to the LiP of P.chrysosporium as being the main decolorizing agent, a recent investigation of thedegradation of selected phthalocyanine dyes and their degradation products showed the

presence of Lac and M nP (Conneely et al., 20 02) and the qualitative analysis of theculture broths helped to propose a pathway for the catalytic process. The findings of Kirby et al. (20 00), demonstrate that Lac is involved in the decolorization of textile dyes by P. tremellosa , however another process must account for the remaining colour removal that is observed in the absence of detectable levels of this enzyme. Lac was theonly one of the three LME detected in supernatants, both in the absence and presence of dye. T. versicolor showed varying decolorizing capacity in different buffers, andsustained repeated additions of individual dyes and dye mixtures in liquid cultures(Swamy and Ramsay, 19 99) . Earlier results from the same group indicated the

Table 6Biochemical features of laccases from newly isolated WRF

Strain T-stability (%) residual pH pH stability (%) MW (kDa) pI (pH)activity at 70 j C opt pH 4 pH 6 monomer dimer

Coriolopsis polyzona CP36

9 2 19.4 396 68 147 5.0, 5.2

Perenniporiaochroleuca PO33

29 2 5.0 1379 63 144 4.5, 5.0

Pycnoporus sanquineus PS6

58 2.5 2.7 45.8 70 4.4, 5.0, 5.3

Pycnoporus sanquineus PS7

70 2.5 0.5 617 65 136 4.3

Perenniporiatephopora PT32

74 2 10.1 638 69 148 5.0, 6.2

Trametes

versicolor TV17

35 2 1.9 48.6 67 4.2, 4.5

Clitocybula dusenii b11 1 2.5 1.2 36.7 66 4.5, 4.7



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involvement of either a mycelial-bound LME or a H 2O2-generating mechanism in thecell wall. Regardless of the MnP and Lac concentrations at the time of dye addition,nitrogen limitation was required for the expression of this activity (Swamy and Ramsay,1999) . LiP was not detected.

Comparisons of the biodegradation of dyes with other recalcitrants may help tounderstand mechanisms. The proposed mechanism of 2,4,6-trinitrotoluene degradation

could also be involved in dye degradation (Stahl and Aust, 1993a,b; Van Aken andAgathos, 2001) . Due to the sequential decolorization of dyes through intermediates of different color, the decolorization has been proposed to be a series of multiple reactions(Vyas and Molitoris, 1995) . However, knowledge is still fragmented, and the funda-mental question remains unanswered: Which are the points of primary attack for decolorization and mineralization (e.g., the azo bond or the aromatic rings)? There isstill a lack of experimental evidence on toxic intermediate accumulation and a variabilityof results among laboratories. Detoxification studies by Heinfling et al. (1997) haveshown that azo dye degradation by B. adusta results in nontoxic compounds. Different

Fig. 5. Enzyme production pattern during decolorization of raw effluenrt. Production of laccase and Mn

peroxidase by T. versicolor during the decolorization of dye-containing effluent. Control experiments were carriedout without effluent ( o ) For decolorization of dye-containing effluent the cultures contained 10% ( . ) and 33%(n ) of raw wastewater in modified Kirk medium.



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specificities between peroxidase isoforms may help to explain variability in reportedresults.

4. Bioreactor systems for decolorization and scale-up

Although a number of publications have appeared on various reactor designs for LME production including stirred tanks, packed beds, airlifts, bubble columns, rotatingdisks, etc., there is a dearth of analogous reports on the use of reactor systemsemploying W RF for waste treatm ent. Towards the design of bioreactor systems for decolorizatio n, Zhang et al. (1999) used an alginate-immobilized basidiomycete, producer of LiP, MnP and Lac, in several reactor configurations. The performanceof continuous packed bed, fedbatch fluidized bed and continuous fluidized bed

bioreactors in terms of decolorization rates and mycelium stability compared favorablyto previously reported continuous decolorization with a fixed film bioreactor usingimmobilized P. chrysospori um (Yang and Yu, 19 96). The adequate mass and oxygentransfer in the fluidised-bed systems ensured reactor stability avoiding excessivemycelial growth. It was suggested that the continuous fluidised-bed system, withlower decolorization rates would be more suitable treating effluents at concentrationsthat are inhibitory to the biomass whereas the fedbatch process would be better for high but not inhibitory dye concentratio ns (Zhang et al., 1999).

The use of rotating biological contactors allows intermittent contact of the myceliumwith the effluent, thus avoiding overgrowth and the problems arising in packed-bedreactors. It was shown that the efficiency of Coriolus (Trametes ) versicolor (a well-knowndecolorizer) in a rotating biodisk contactor varied depending on the biofilm thickness,rotational speed and carbon source concentrati on (Kapdan and Kargi, 20 02). However,there is room for optimization of such systems.

Anaerobic decolorization of azo and other water-soluble dyes is possible viaoxidation reduction reaction with hydrogen, yielding methane and hydrogen sulphide(Carliell et al., 19 96). An additional carbon source is required as an electron donor so that electrons released are cascaded down to the final electron acceptor, the dye. The reuse of the accompanying biogas could reduce the energy costs of such application; however, the price of glucose may be a limiting factor in scale-up proje cts (Robinson et al., 200 1a).Moreover, the toxic amines that are generated when azo and nitro compounds are reduced(Banat et al., 1996; Beydilli et al., 19 98), may create an additional pollution problem for waterways.

The exposure of the reduced azo and nitro compounds to oxygen could causereappearance of coloration, however aromatic amines can be mineralized by nonspecificenzymes through hydroxylation and ring-opening of the aromatic compounds under activated sludge treatment. Coupled anaerobicaerobic degradation of dyes was tested byONeill et al. (20 00), by means of HPLC-UV analyses. It was verified that toxic amino-containing compounds were formed during the anaerobic stage of an Upflow AnaerobicSludge Blanket reactor for treatment of simulated textile effluent. During the succeedingaerobic stage, some degradation of nitrogen-containing aromatic derivatives took placewith mineralization of organic nitrog en (ONeill et al., 20 00). Heat-treatment liquor of



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hydrolases but without compromising performance requirements (e.g., durability of color on textile fibers).

6. Concluding remarks

The varying methods to assess decolourization are also a perplexing factor towardsdeveloping strategies for bioremediation. HPLC analyses for individual dyes are possiblein some cases, though this is labour intensive and probably not applicable for monitoringcomplex transformations. Further studies should be conducted, using advanced analyticaltechniques, to elucidate the catabolic processes involved in the degradation of distinct dyegroups by the LME of WRF. In the near future, the progress in the field of nanotechnologycould provide biochemical engineers powerful tools for studying cell surface and topology

to better understand the importance of membrane-bound oxidoreductases and their role ingrowth-associated degradation of organic dyes by WRF.

Acknowledgements

Financial support by the Directorate General for Technology, Research and Energy of the Walloon Regional Government of Belgium through its BIOVAL program (grant no981/3870) is gratefully acknowledged. The collaboration of S. Vanhulle, F. Buchon, M.Lucas, S. Caillou, V. Mertens and A.-M. Corbisier is also acknowledged.

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