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Biotechnology Advances 22 (2003) 161 187

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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 directly
involved in the degradation of various xenobiotic compounds and dyes. This review summarizes the
state of the art in the research and prospective use of WRF and their enzymes (lignin-modifying
enzymes, 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 its
colored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks to
emerging knowledge of the physiology of these organisms as well as of the biocatalysis and stability
characteristics 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 commercially
successful synthetic dye. By the end of the 19th century, ten thousand new synthetic
dyes had been developed and manufactured. Nowadays, India, the former USSR,

* Corresponding author. Tel.: +32-10-47-3644; fax: +32-10-47-3062.


E-mail address: agathos@gebi.ucl.ac.be (S.N. Agathos).
0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2003.08.011

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

Eastern Europe, China, South Korea and Taiwan consume approximately 600 thousand
tons (kt) of dyes per annum (Ishikawa et al., 2000). Since 1995, China has been the
leading producer of dyestuffs, exceeding 200 kt per annum (Ishikawa et al., 2000). The
total annual world textile dye production is estimated at about 800 kt (Zollinger, 1991).
In 1999 the value of the global dyestuff market was estimated at 6.6 billion US$,
North America accounting for 1.2 billion US$, Central and South America for 0.7
billion US$, Western Europe for 1.2 billion US$ and Asia for 2.7 billion US$ (Will et
al., 2000) The distribution of global dyestuff market has changed during the last
decade, with Asia being the largest dyestuff market today (about 42%). Even though
the dye industry is characterized by a large number of producers (about 2000
worldwide), just four Western companies accounted for nearly half of the market in 2000
(Will et al., 2000).
Dyestuffs can be classified according to origin, chemical and/or physical properties
and characteristics related to the application process. A division into native and
synthetic dyes is inadequate, since nowadays the synthesis of many natural substances
is possible. A classification into textile, leather, paper or food dyes gives only a clue
as to the characteristics of the colorant. A more suitable categorization for the
applications sector should be based upon the modern dyeing technologies (e.g., inks,
disperse dyes, pigments or vat dyes). A systematic classification of dyes according to
chemical structure is the color index (C.I., Table 1). This scheme is also useful for
estimating the possible biodegradability of dyes. A listing of synthetic dyes according
to their most predominant chemical structures is given in Table 2.
All dyes used in the textile industry are designed to resist fading upon exposure to
sweat, light, water, many chemicals including oxidizing agents, and microbial attack.
During processing, up to 15% of the used dyestuff are released into the process water
(Vaidya and Datye, 1982). Dye-containing effluents are hardly decolorized by conventional biological wastewater treatments (Shaul et al., 1991; Willmott et al., 1998). In
addition to their visual effect and their adverse impact in terms of chemical oxygen
demand, many synthetic dyes are toxic, mutagenic and carcinogenic (Michaels and
Lewis, 1985; Chung et al., 1992). Moreover, the frequently high volumetric rate of

Table 1
Classes of synthetic dyes according to color index (C.I.)
Code

Chemical class

Code

Chemical class

Code

Chemical class

10,000
10,300
11,000
20,000
30,000
35,000
37,000
40,000
40,800
41,000

Nitroso
Nitro
Monoazo
Disazo
Trisazo
Polyazo
Azoic
Stilbene
Carotenoid
Diphenylmethane

42,000
45,000
46,000
47,000
48,000
49,000
49,400
50,000
51,000
52,000

Triarylmethane
Xanthene
Acridine
Quinoline
Methine
Thiazole
Indamine/Indophenol
Azine
Oxazine
Thiazine

53,000
55,000
56,000
57,000
58,000
73,000
74,000
75,000
76,000
77,000

Sulfur
Lactone
Aminoketone
Hydroxyketone
Anthraquinone
Indigoid
Phthalocyanine
Natural
Oxidation Base
Inorganic

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

163

Table 2
Classes of organic dyes: structures of representative colorants

industrial effluent discharge in combination with increasingly stringent legislation, make


the search for appropriate treatment technologies an important priority (ONeill et al.,
1999). Abiotic means of reduction of azo and other dyes exist but require highly
expensive catalysts and reagents (Robinson et al., 2001a). A number of biotechnological
approaches have been suggested by recent research as of potential interest towards
combating this pollution source in an ecoefficient manner, including the use of bacteria
or 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). These
constitute a diverse ecophysiological group comprising mostly basidiomycetous (and,
to a lesser extent, litter-decomposing) fungi capable of extensive aerobic lignin
depolymerization and mineralization. This property is based on the WRFs capacity to
produce one or more extracellular lignin-modifying enzymes (LME), which, thanks to

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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 and
laccases (Lac), E.C. 1.10.3.2, are directly involved not only in the degradation of
lignin in their natural lignocellulosic substrates (Becker and Sinitsyn, 1993; Hatakka,
1994) but also in the degradation of various xenobiotic compounds (Barr and Aust,
1994; Pointing, 2001; Scheibner et al., 1997) including dyes (Glenn and Gold, 1983;
Pasti-Grigsby et al., 1992; Paszczynski et al., 1992; Spadaro et al., 1992). Some white
rot fungi produce all three LME while others produce only one or two of them
(Hatakka, 1994). LME are essential for lignin degradation, however for lignin
mineralization they often combine with other processes involving additional enzymes.
Such auxiliary enzymes (by themselves unable to degrade lignin) are glyoxal oxidase
and superoxide dismutase for intracellular production of H2O2, a cosubstrate of LiP
and MnP, as well as glucose oxidase, aryl alcohol oxidase and cellobiose dehydrogenase involved in feedback circuits and linking ligninolysis with cellulose and
hemicellulose degradation in nature (Leonowicz et al., 1999). 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 fatty
acids (Kapich et al., 1999) or thiols (Hofrichter et al., 1998a). In this way, an older
concept of ligninolysis reemerges, enzymatic combustion (Kirk and Farrell, 1987).
By extension, this enzyme-assisted process is applicable to the degradation of many
other recalcitrant molecules, such as synthetic dyes. Current views of LME are given
in two recent reviews with emphasis on Lac (Leonowicz et al., 2001) or MnP
(Hofrichter, 2002).
The main LME are oxidoreductases, i.e., two types of peroxidases, LiP and MnP
(Fig. 1), and a phenoloxidase, Lac (Fig. 2). The physiology of LME production by
WRF 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 experimental
conditions reported: LME are produced by WRF during their secondary metabolism
since 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); production
of LiP and MnP is generally optimal at high oxygen tension but is repressed by
agitation in submerged WRF liquid culture, while Lac production is often enhanced by
agitation; frequently, more than one isoforms of LME are expressed by different taxa
and culture conditions. These features are important in the process design and
optimization of fungal treatment of colorant-containing effluents.

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

165

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

The most common ligninolytic peroxidases produced by almost all white-rot basidiomycetes and by various litter-decomposing fungi are manganese peroxidases (MnP).
These are glycosylated glycoproteins (Nie et al., 1999) with an iron protoporphyrin IX
(heme) prosthetic group (Glenn and Gold, 1985), molecular weights between 32 and 62.5
kDa (Hofrichter, 2002) and are secreted in multiple isoforms (Leisola et al., 1987; Urzua et
al., 1995). MnP preferentially oxidize Mn2 + into Mn3 + (Glenn et al., 1986), which is
stabilized by chelators such as oxalic acid (Wariishi et al., 1992), itself also excreted by the
fungi (Galkin et al., 1998; Kuan and Tien, 1993; Takao, 1965). Chelated Mn3 + acts as a
highly reactive (up to 1510 mV in H2O, Cui and Dolphin, 1990) low molecular weight,
diffusible redox-mediator. Thus, MnP are able to oxidize and depolymerize their natural

Fig. 2. The catalytic cycle of laccases.

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substrate, i.e., lignin as well as recalcitrant xenobiotics such as nitroaminotoluenes


(Scheibner et al., 1997; Van Aken et al., 1999) and textile dyes (Heinfling et al.,
1998a). In vitro depolymerization can be enhanced in the presence of cooxidants such
as thiols (e.g., glutathione) or unsaturated fatty acids (e.g., Tween 80) (Hofrichter, 2002).
Lignin peroxidases (LiP) catalyze the oxidation of nonphenolic aromatic lignin
moieties and similar compounds. LiP are well known as part of the ligninolytic system
both of aphyllophoralic and agaricalic fungi (Glenn et al., 1983; Hatakka et al., 1987;
Hofrichter and Fritsche, 1997). The extracellular N-glycosylated LiP with molecular
masses between 38 and 47 kDa contain heme in the active site and show a classical
peroxidase mechanism (Tien et al., 1986, Fig. 1). LiP catalyze several oxidations in the
side chains of lignin and related compounds (Tien and Kirk, 1983) by one-electron
abstraction to form reactive radicals (Kersten et al., 1985). Also the cleavage of aromatic
ring structures has been reported (Umezawa and Higuchi, 1987). The role of LiP in
ligninolysis could be the further transformation of lignin fragments which are initially
released by MnP. LiP are not essential for the attack on lignin: several highly active WRF
and litter-decaying fungi (e.g., Ceriopsis subvermispora, Dichotomitus squalens, Panus
tigrinus, Rigidosporus lignosus) do not excrete this enzyme (Galliano et al., 1991;
Hatakka, 1994; Maltseva et al., 1991; Perie and Gold, 1991). LiP have been used to
mineralize a variety of recalcitrant aromatic compounds, such as three- and four-ring PAHs
(Gunther et al., 1998), polychlorinated biphenyls (Krcmar and Ulrich, 1998) and dyes
(Chivukula et al., 1995). 2-Chloro-1,4-dimethoxybenzene, a natural metabolite of WRF is
reported to act as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al., 1998).
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 only
Mn2 + but also phenolic and nonphenolic aromatic compounds including dyes. VP have
been described in species of Pleurotus and Bjerkandera (Heinfling et al., 1998a,b; Mester
and Field, 1998). A comprehensive review of the molecular biology of WRF peroxidases
is given by Martnez (2002) and by Conesa et al. (2002).
Fungal laccases as part of the ligninolytic enzyme system are produced by almost all
wood- and litter-transforming basidiomycetes. This group of N-glycosylated extracellular
blue oxidases with molecular masses of 60 390 kDa (Call and Mucke, 1997; Reinhammar, 1984), contain four copper atoms in the active site (as Cu2 + in the resting enzyme)
that are distributed among different binding sites, and are classified into three types with
differential specific characteristic properties (McGuirl and Dooley, 1999; Messerschmidt,
1997). Laccases catalyze the oxidation of a variety of aromatic hydrogen donors with the
concomitant reduction of oxygen to water (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 focus
on their industrial applicability (Bajpai, 1999; Gianfreda et al., 1999; Rodrguez et al.,
1999; Yaropolov et al., 1994), molecular genetics (Cullen, 1997; Karahanian et al., 1998;
Ong et al., 1997; Collins and Dobson, 1997) and cloning (Hatamoto et al., 1999). Laccases
have been reported to oxidize many recalcitrant substances, such as chlorophenols (Fahr et
al., 1999; Grey et al., 1998; Ricotta et al., 1996; Roy-Arcand and Archibald, 1991), PAHs
(Majcherczyk et al., 1998), lignin-related structures (Bourbonnais et al., 1996; Boyle et al.,
1992), organophosphorous compounds (Amitai et al., 1998), nonphenolic lignin model

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

167

compounds (Kawai et al., 1988; Majcherczyk et al., 1999), phenols (Bollag et al., 1988;
Xu, 1996) and last but not least, aromatic dyes (Abadulla et al., 2000; Chivukula and
Renganathan, 1995; Rodrguez et al., 1999). A comparative summary of the main
characteristics of LME from WRF is given in Table 3.
2.2. Small-molecule mediators
Given the random polymer nature of lignin and the bulk of LME, direct and specific
interactions between lignin (or recalcitrant structural analogs) and LME are highly

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

MnP 1.11.1.13

LiP 1.11.1.14

Lac 1.10.3.2

diarylpropan O2, H2O2


oxidoreductases
Heme

p-benzendiol: O2-oxidoreductases

Prosthetic group

Mn(II): H2O2
oxidoreductases
Heme

38 47
N
monomers; up to 15

pI
pH range
E0 (mV)
C C cleavage
H2O2-regulated
Stability
Native mediators
Specificity

32a 62.5b (122a)


N
monomers;
up to 11d
2.8e 7.2f
2.6g 4.5h
1510i
yes
yes
+++
Mn2 +; Mn3 +
Mn2 +

Secondary and
synthetic mediators

Thiols, unsaturated
fatty acids

MW (kDa)
Glycosylation
Isoforms

3.2 4.7
2.0 5.0
1450j
yes
yes
+
VA?l, 2Cl-14DMBm
broad, aromatics,
incl. nonphenolics
NO

1 type-1-Cu, 1 type-2-Cu,
2 coupled type-3-Cu,
59 110 (tetramers V 390c
N
mono-, di-, tetramers; several
2.6 4.5
2.0 8.5
500 800k
no
no
+++
3-HAAn
broad, phenolics
ABTSo, 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 H2O (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: 2,2V-azinobis(3-ethylbenzthiazoline-6-sulfonate); HBT:1-hydroxybenzotriazole (Bourbonnais et al.,
1996).

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

improbable (Evans and Hedger, 2001). Rather low-molecular weight, diffusible redox
mediators provide high redox potentials (>900 mV) to attack lignin and are able to
migrate into the lignocellulose complex. Examples of native as well as synthetic
mediators are given in Table 4. They could be involved in the LME-catalyzed generation
of reactive radical moieties from a variety of lignin-like substrates, but also in the
formation of reactive oxygen species (ROS) which either directly or indirectly could
attack lignin or xenobiotic molecules (Hammel, 1996; Van Aken and Agathos, 2001,
2002).
Organic acids, excreted by several fungal organisms, chelate and stabilize Mn3 +. MnP
was found to simultaneously decompose organic acids (such as malonate) oxidatively and
oxidize Mn2 + to Mn3 + even in the absence of H2O2. Thus, organic acids are postulated to
S
be the origin of carbon-centered radicals (acetic acid radicals, COOH C H2, Reaction 1),
S
S
peroxyl radicals (COOH CH2OO , Reaction 2), superoxide (O2 , Reactions 5 and 8),
S
formate radicals (CO2 , Reactions 6 and 7). Such radicals could be a source of peroxides,
which can be used by MnP as substrates instead of H2O2. Consequently, even fungi

Table 4
Native and synthetic mediators in LME systems
Mediator
Native mediators
Mn3 +
Organic acids
(malonate, oxalate, etc.)

Veratryl alcohol
3-Hydroxyanthranilic acid
(3-HAA)
2-Chloro-1,4dimethoxybenzene
(2Cl-14DMB)
Synthetic mediators
1-Hydroxybenzotriazole
(1-HBT)

Violuric acid

2,2V-Azinobis
(3-ethylbenzthiazoline6-sulfonate) (ABTS)

Organism (enzyme)

Reference

Phanerochaete
chrysosporium (MnP)
Armillaria mellea,
Fomes annosus, Pleurotus ostreatus,
Phanerochaete chrysosporium,
Phlebia radiata, Cenporiopsis
subvermispora, Nematoloma
frowardii (LiP, MnP)
Phanerochaete chrysosporium (LiP)
Pycnoporus cinnabarinus (Lac)

Wariishi et al., 1992

Trametes versicolor (LiP)

Trametes versicolor, Trametes


villosa, Pycnoporus cinnabarinus,
Botrytis cinerea, Myceliophthora
thermophila, Coriolopsis gallica,
Pleurotus ostreatus various
organisms (Lac)
Trametes villosa, Pycnoporus
cinnabarinus, Botrytis cinerea,
Myceliophthora thermophila (Lac)
Trametes versicolor, Coriolopsis
gallica, Pleurotus ostreatus,
various organisms (Lac)

Galkin et al., 1998;


Hofrichter et al., 1999;
Takao, 1965

Lundquist and Kirk, 1978


Eggert et al., 1996;
Eggert et al., 1997
Teunissen and Field, 1998

Bourbonnais et al., 1996;


Crestini and Argyropoulos,
1998; Li et al., 1999;
Pickard et al., 1999

Li et al., 1999

Bourbonnais et al., 1996;


Crestini and Argyropoulos,
1998; Pickard et al., 1999

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

169

obviously lacking H 2O 2 -generating oxidases could be efficient lignin-degraders


(Hofrichter et al., 1998b) and, by extension, useful in the degradation of xenobiotics such
as dyes.
S

COOH  CH2  COOH Mn3 ! COOH  C H2 CO2 H Mn2


Reaction 1
S

COOH  C H2 O2 ! COOH  CH2 OO

Reaction 2

COOH  CH2 OO Mn2 ! COOH  CH2 OOH Mn3

Reaction 3

COOH  CH2 OOH 2 Mn2 ! COOH  CHO H2 O 2 Mn3

Reaction 4

Reaction 5

Reaction 6

COOH  CH2 OO O2 ! COOH  COOH O2 H


COOH  CHO Mn3 1=2 O2 ! HCOOH CO2 Mn2
S

COOH  COOH Mn3 ! CO2 CO2 Mn2


S

CO2 O2 ! CO2 O2


S

O2 Mn2 2 H ! H2 O2 Mn3


H2 O2 2 Mn2 ! H2 O 2 Mn3

Reaction 7

Reaction 8

Reaction 9

Reaction 10

On the other hand organic acids (e.g., oxalate) chelate cations including Fe2 + (Dutton et
al., 1993), therefore such acids are indirectly involved in the regulation of Fentons
reaction due to regulation of Fe2 + concentration (Fenton, 1894; Koenigs, 1972), which
supplies fungal degradation reactions with hydronium ions (H3O+) and hydroxyl radicals
(HOS, HO). Recent evidence strongly suggests the involvement of formyl and superoxide
free radicals in the in vitro mineralization of recalcitrant nitroaminoaromatic molecules by
MnP or by its biomimetic analog Mn(III)/oxalate/O2 (Van Aken and Agathos, 2002).
The in vitro degradation of lignin and other recalcitrant molecules by MnPs is
considerably enhanced in the presence of thiols [reduced glutathione (GSH), cysteine

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

(Cys)]. Thiols were shown to promote the attack of the aromatic ring of veratryl alcohol
and nonphenolic h O 4 lignin model dimers (DAnnibale et al., 1996; Forrester et al.,
1988; Wariishi et al., 1989). Although fungal secretion of reduced thiols is unlikely, thiolic
peptides released during partial cell lysis might be a source of thiol mediators (Hofrichter,
2002). Both MnP and chelated Mn(III) catalyze the oxidation of GSH to reactive free
glutathionyl radical GS, whose production correlates with the mineralization of recalcitrant
aminonitroaromatic compounds (Van Aken et al., 2000a,b).
Veratryl alcohol (VA, 3,4-dimethoxy benzyl alcohol), a secondary metabolite of several
WRF (de Jong et al., 1994), after its oxidation to the VA cation radical (VA+) by LiP, acts
as a mediator for the degradation of lignin (Farrell et al., 1989; Tien and Kirk, 1983).
However, due to the short life span of VA+ long-distance charge transfers are not likely to
occur. Mediating properties of VA could be enhanced if the radical is somehow complexed
to the LiP (Lundell, 1993). Nevertheless, LiP is stimulated by VA probably by protecting
the enzyme against the damaging effect of H2O2 (Akhtar et al., 1997).
3-Hydroxyanthranilic acid (3-HAA) was the first natural mediator for laccases
described. This mediator enables a laccase-catalyzed oxidation of nonphenolic lignin
model dimers (Eggert et al., 1996). To delignify kraft pulp by laccase a number of
synthetic mediators have been tested. For instance, using 2,2V-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) laccases are able to attack nonphenolic lignin model compounds and to delignify kraft pulp (Bourbonnais et al., 1995). The discovery of 1hydroxybenzotriazole (HBT), an effective laccase mediator in pulp processing (Call, 1994)
lead to a new class of mediators with NOH as the functional group, which is oxidized to a
reactive radical (R NO). These mediators [e.g., 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid (HNNS), 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), and Remazol
brilliant blue (RBB)] have been shown to support delignification reactions by laccases
(Bourbonnais et al., 1997).

3. Decolorization of textile dyes and effluents by WRF


WRF are the most intensively studied dye-decolorizing microorganisms. As stated
earlier, thanks to their nonspecific LME these fungi are able to transform a wide range of
organic compounds. Primarily, the decolorization of sulfonated polymeric dyes was used
to assay ligninolytic activities (Glenn and Gold, 1983; Freitag and Morrell, 1992) and to
assess the biodegradation capabilities of WRF (Field et al., 1993; Cripps et al., 1990).
Later on, numerous WRF strains were used for the decolorization of distinct synthetic
(textile) dyes and synthetic effluents, i.e., dye mixtures (Jarosz-Wilkolazka et al., 2002;
McMullan et al., 2001). Table 5, without claims to comprehensiveness, summarizes the
existing literature regarding textile dye decolorization by WRF, with an effort to indicate,
wherever possible, the enzymes involved. Uptake effects or dye sorption by WRF mycelia
without real degradation are generally minimal (Glenn and Gold, 1983). These effects are,
rather, seen in applications of non-WRF, such as Aspergillus niger, whose (dead) biomass
could be used as an adsorbent (Fu and Viraraghavan, 2000; Sumathi and Manju, 2000) and
serve as part of a technical solution in water pollution control. The involvement of
individual LME in decolorization has been confirmed using in vitro (cell-free) LME

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

171

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

LMEa (Reference)

Dyeb (Reference)

Bjerkandera adusta

 |LiP|MnP
(Kaal et al., 1995)

Bjerkandera sp.

 |  |MnP
(Moreira et al., 2000)

Reactive Orange 96N = N, Reactive Violet 5N = N,


Reactive Black 5N = N, Reactive Blue 15PC, Reactive
Blue 38PC (Heinfling et al., 1997)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Reactive Violet 5N = N, Reactive Black 5N = N, Reactive
Blue 38PC by MnP (Heinfling et al., 1998a,b)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Brilliant GreenTPM, Cresol RedTPM, Congo RedN = N
(Gill et al., 2002)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Brilliant GreenTPM, Cresol RedTPM, Crystal VioletTPM,
Congo RedN = N, Orange IIN = N (Gill et al., 2002)
Reactive Blue 5PAQ (Kim and Shoda, 1999)

Ceriporia
metamorphosa
Daedalea flavida
Daedaleopsis
confragosa
Dichomitus squalens
Geotrichum
candidum
Irpex lacteus

Lentinus tigrinus

ND

Lac|  |MnP (Perie


and Gold, 1991)
(Kim and Kim, 1995 )
Lac|LiP|MnP
(Novotny et al., 2000)

Lac|  |MnP (Moreira


et al., 2000)

Mycoacia nothofagi
Phanerochaete
chrysosporium

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

Lac|  |MnP (Chagas


and Durrant, 2001)

Methyl redN = N, Congo RedN = N, Naphtol Blue


BlackN = N, Remazol Brilliant Blue RPAQ, Bromophenol
blueTPM, Copper (II) phthalocyaninetetrasulfonic acid
tetrasodium saltMC, Poly R-478PAQ (Novotny et al., 2001)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Remazol Turquoise BluePC (Conneely et al., 1999)
azo dyes, Azure Blue, Cresol RedTPM, Crystal
VioletTPM, Bromophenol blueTPM (Cameron et al., 2000)
Acid Green 27PAQ, Copper phtalocyanine tetrasulphonic
acid tetrasodium saltMC, Indigo Carmine, Neutral
RedHC, Acid Red 106N = N, Mordant Yellow 10N = N,
Brilliant YellowN = N, ChrysophenineN = N, Chlorazol
YellowN = N, Cibacron Brilliant Yellow 3G-P (Reactive
Yellow 2)N = N;HC, Cibacron Brilliant Red 3B A
(Reactive Red 4)N = N;HC, Orange IIN = N, Crystal
VioletTPM, Brilliant GreenTPM (Knapp et al., 1995)
Remazol Brilliant Blue RPAQ (Novotny et al., 2001)
Indigo (Balan and Monteiro, 2001)
AmaranthN = N, New CoccineN = N, Orange GN = N,
TartrazineN = N (Chagas and Durrant, 2001)
Reactofix OrangeN = N, Reactofix Golden YellowN = N,
Reactofix Blue HE2RN = N, Navilene BlackN = N, Sulphur
GreenN = N, Sulphur RedN = N, Navione BlueN = N, Vat
BrownN = N (Capalash and Sharma, 1992)
Red 2BAB (Jain et al., 2000)
Congo RedN = N (Gill et al., 2002)
(continued on next page)

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

Table 5 (continued)
Organism

LMEa (Reference)

Phanerochaete
sordida
Phellinus gilvus
Phellinus
pseudopunctatus
Phlebia brevispora

 |  |MnP (Moreira
et al., 2000)

Phlebia (Merulius)
tremellosa

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

Phlebia fascicularia
Phlebia floridensis
Phlebia radiata

Lac|  |MnP (Moreira


et al., 2000)

Piptoporus betulinus

Pleurotus eryngii

Pleurotus ostreatus

Pleurotus sajor-caju

 |LiP|MnP (Martnez et al., 1996; Heinfling et al., 1998a,b)


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

Lac|  | (Chagas and


Durrant, 2001)
 |  |MnP? (Boyle
et al., 1992)

Dyeb (Reference)
Reactive Orange 96N = N, Reactive Violet 5N = N,
Reactive Black 5N = N, Reactive Blue 15PC, Reactive
Blue 38PC (Heinfling et al., 1997)
Everzol Turquoise Blue GPC, Everzol Yellow 4GL,
Everzol Red RBN, Orange K-GL, Everdirect Supra
Yellow PG (Kapdan et al., 2000a)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Indigo (Balan and Monteiro, 2001)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Brilliant GreenTPM, Cresol RedTPM, Crystal VioletTPM
(Gill et al., 2002)
Cibacron Red, Remazol Navy Blue, Remazol Red,
Cibacron Orange, Remazol Golden Yellow, Remazol
Blue, Remazol Turquoise Blue, Remazol Black B,
Mixture (Kirby et al., 2000)
Brilliant GreenTPM, Cresol RedTPM, Crystal VioletTPM,
Congo RedN = N, Orange IIN = N (Gill et al., 2002)
Brilliant GreenTPM, Cresol RedTPM, Crystal VioletTPM,
Congo RedN = N, Orange IIN = N (Gill et al., 2002)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Crystal VioletTPM, Congo RedN = N, Orange IIN = N (Gill
et al., 2002)
Acid Green 27PAQ, Copper phtalocyanine tetrasulphonic
acid tetrasodium saltMC, Indigo Carmine, Acid Red
106N = N, Brilliant YellowN = N, ChrysophenineN = N,
Chlorazol YellowN = N, Cibacron Brilliant Yellow 3G-P
(Reactive Yellow 2)N = N;HC, Cibacron Brilliant Red 3B
A (Reactive Red 4)N = N;HC, Orange IIN = N, Crystal
VioletTPM, Brilliant GreenTPM (Knapp et al., 1995)
Reactive Violet 5N = N, Reactive Black 5N = N, Reactive
Blue 38PC by MnP (Heinfling et al., 1998a,b)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Acid Green 27PAQ, Copper phtalocyanine tetrasulphonic
acid tetrasodium saltMC, Indigo Carmine, Neutral
RedHC, Mordant Yellow 10N = N, Brilliant YellowN = N,
ChrysophenineN = N, Cibacron Brilliant Yellow 3G-P
(Reactive Yellow 2)N = N; HC, Cibacron Brilliant Red 3B
A (Reactive Red 4)N = N;HC, Orange IIN = N, Crystal
VioletTPM, Brilliant GreenTPM (Knapp et al., 1995)
AmaranthN = N, New CoccineN = N, Orange GN = N,
TartrazineN = N (Chagas and Durrant, 2001)
Indigo (Balan and Monteiro, 2001)

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

173

Table 5 (continued)
Organism

LMEa (Reference)

Dyeb (Reference)

Polyporus ciliatus

Lac|  |MnP (Moreira


et al., 2000)

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


(Moreira et al., 2000)
Brilliant GreenTPM, Crystal VioletTPM, Congo RedN = N
Brilliant GreenTPM, Crystal VioletTPM, Congo RedN = N
(Gill et al., 2002)
Orange GN = N, AmaranthN = N, Bromophenol BlueTPM
Malachite GreenTPM (Pointing and Vrijmoed, 2000)
Indigo (Balan and Monteiro, 2001)
Orange IIN = N, Reactive Blue 38PC, Poly R-478PAQ
(Moreira et al., 2000)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)
Everzol Turquoise Blue GPC, Everzol Yellow 4GL,
Everzol Red RBN, Orange K-GL, Everdirect Supra
Yellow PG (Kapdan et al., 2000a,b)
Acid Green 27PAQ, Copper phtalocyanine tetrasulphonic
acid tetrasodium saltMC, Indigo Carmine, Neutral
RedHC, Acid Red 106N = N, Mordant Yellow 10N = N,
Brilliant YellowN = N, ChrysophenineN = N, Chlorazol
YellowN = N, Cibacron Brilliant Yellow 3G-P (Reactive
Yellow 2)N = N; HC, Cibacron Brilliant Red 3B A
(Reactive Red 4)N = N; HC, Orange IIN = N, Crystal
VioletTPM, Brilliant GreenTPM (Knapp et al., 1995)
AmaranthN = N, Remazol BlackN = N, Remazol Brilliant
BluePAQ, Reactive Blue 15PC, TropaeolinN = N, Remazol
OrangeN = N, Remazol Brilliant Red BBN = N (Swamy
and Ramsay, 1999)
Reactive Orange 96 N = N, Reactive Violet 5 N = N,
Reactive Black 5 N = N, Reactive Blue 15PC, Reactive
Blue 38PC (Heinfling et al., 1997)
Remazol Brilliant Blue RPAQ, Poly R-478PAQ (Novotny
et al., 2001)

Polyporus
sanguineus
Pycnoporus
sanguineus
Stereum hirsutum

Lac|  |MnP (Moreira


et al., 2000)

Stereum rugosum
Trametes (Coriolus)
versicolor

Lac|LiP|MnP
(Hatakka, 1994)

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

PAQ

systems from WRF culture supernatants. LME-producing profiles vary. For instance, Lac
was the main enzyme involved in dye decolorization by cultures of Phlebia tremellosa
(Kirby et al., 2000; Robinson et al., 2001b) and by Pleurotus sajorcaju (Chagas and
Durrant, 2001), whereas LiP or MnP activity was absent (Kirby et al., 2000). MnP could
only be detected when the culture medium was supplemented with MnCl2. Elsewhere, the
presence of LiP and/or MnP in addition to Lac (Pleurotus ostreatus, Schizophyllum
commune, Sclerotium rolfsii, Neurospora crassa) seemed to increase by up to 25% the
degree of decolorization of individual commercial triarylmethane, anthraquinonic, and
indigoid textile dyes using enzyme preparations (Abadulla et al., 2000). On the contrary,
MnP was reported as the main enzyme involved in dye decolorization by Phanerochaete
chrysosporium (Chagas and Durrant, 2001) and LiP for Bjerkandera adusta (Robinson et

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

al., 2001b). LiP was also considered as the principal decolorizing enzyme in cultures of P.
chrysosporium (Kirby et al., 1995). It is clear that LME play significant roles in dye
metabolism by WRF (McMullan et al., 2001). This is not surprising, given the structural
similarity of most commercially important dyes (Table 2) to lignin (sub)structures
amenable to transformation by LME, as described in the preceding sections. In vitro
decolorizations using LME were examined, e.g., using Lac from Pyricularia oryzae
(Chivukula and Renganathan, 1995) and Trametes versicolor (Wesenberg et al., in
preparation), LiP from P. chrysosporium (Chivukula et al., 1995; Heikkila et al., 1998)
and MnP (or, more accurately, VP) from B. adusta and Pleurotus eryngii (Heinfling et al.,
1998a,b). Of most interest for practical applications appears to be the different enzymatic
pattern depending on the ligninolytic strains used (McMullan et al., 2001). Dye
mineralization by WRF was confirmed by 14C-ring-labeled azo dyes that were mineralized
using P. chrysosporium (Spadaro et al., 1992). The influence or not of the substitution
pattern on the dye mineralization rates is a matter of controversy (Paszczynski et al., 1992;
Spadaro et al., 1992), though it is clear that dye decolorization is not equivalent to dye
mineralization. There is a definite gap in our current knowledge of decolorization and,
even more so, of mineralization mechanisms. With a lack of insight concerning potentially
toxic albeit colorless accumulating intermediates, our capacity to evaluate the true
technical potential of WRF and their LME remains incomplete.
However, these difficulties are even greater if one considers that complex mixed
effluents are extremely variable in composition in one and the same factory, as is often
the case in the textile industry. Thus, the decolorization of real effluents requires an
appropriate choice of fungal strain as well as of reactor environment. Real textile dye
effluents contain not only dyes but also salts, sometimes at very high ionic strength and
extreme pH values, chelating agents, precursors, by-products, surfactants, etc. As
reported in a systematic study by Knapp et al. (1995), 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 as
well as anionic detergents, which could be found in real textile industrial effluents,
inhibited Polyporus sp. and Trametes villosa up to 20%, whereas S. commune LMEs
were inhibited up to 70% (Abadulla et al., 2000). Thus, in spite of the high
decolorization efficiency of some strains, decolorizing a real industrial effluent is quite
troublesome. Knapp and Newby (1999) were the first to report the decolorization of an
effluent of the chemical industry containing an azo-chromophore by WRF. Only
recently the first attempt to apply WRF to decolorize a real textile dye industrial
effluent was published by our group. Using the agaric white-rot fungus, Clitocybula
dusenii maximal decolorization rates were achieved over a period of 20 days at 28 jC
using fourfold diluted dye-containing effluent (6559 color units as defined in Standard
Methods (Clesceri et al., 1998)) on a 5-day pregrown mycelium (Wesenberg et al.,
2002) (Fig. 3). The main enzyme involved in the decolorization achieved by C. dusenii
was 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, MnP
appeared to be induced at higher effluent concentrations and might also have a role in

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

175

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) based
on a reference to K2PtCl6 as well as on the measured absorbance as derived from the integrated surface area of
spectral scans (400 700 nm; empty symbols, right-hand axis). The cultures contained 10% (./o), 25% (E/4)
or 33% (n/5) of raw wastewater in modified Kirk medium (Wesenberg et al., 2002).

the decolorization by C. dusenii (Wesenberg et al., 2002). The extraction of these


enzymes allowed a first investigation of biochemical features of the Lac of several new
dye effluent-decolorizing WRF (Table 6).
The inherent complexity of both the dyes structures and the enzymatic transformation
mechanisms makes the elucidation of the degradation pathways a difficult task. Possible
non-LME degrading mechanisms have been suggested (Pasti and Crawford, 1991) and dye
degradation-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. 4. Typical profile of laccase production by WRF. Laccase activity during culture of Trametes versicolor with
a draw-fill method. When the laccase activity (..) reached its maximum, the culture liquid was harvested and
replaced by fresh medium (- - - - -).

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

Table 6
Biochemical features of laccases from newly isolated WRF
Strain

Coriolopsis
polyzona CP36
Perenniporia
ochroleuca PO33
Pycnoporus
sanquineus PS6
Pycnoporus
sanquineus PS7
Perenniporia
tephopora PT32
Trametes
versicolor TV17
Clitocybula dusenii b11

T-stability (%) residual


activity at 70 jC

pH
opt

pH stability (%)
pH 4

pH 6

monomer

dimer

19.4

396

68

147

5.0, 5.2

29

5.0

1379

63

144

4.5, 5.0

58

2.5

2.7

70

70

2.5

0.5

617

65

136

4.3

74

10.1

638

69

148

5.0, 6.2

35

1.9

48.6

67

4.2, 4.5

2.5

1.2

36.7

66

4.5, 4.7

45.8

MW (kDa)

pI (pH)

4.4, 5.0, 5.3

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. (2002),
suggesting a differential inducing effect of the effluent on the LME production pattern
(Fig. 5). An earlier study of Wong and Yu (1999) proposed a mechanism for the
increased decolorization capacity of T. versicolor Lac, that involves the decolorization
of nonsubstrate dyes in effluents via substrate dyes which also act as mediators in the
Lac 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 lignintransforming actinomycete Streptomyces chromofuscus is a weak decolorizer compared
to P. chrysosporium (Paszczynski et al., 1992), whose decolorizing capacity is due to
LiP and not MnP (Young and Jian, 1997). Immunochemical methods have revealed that
a fraction of the LiP produced by P. chrysosporium remains associated with the fungal
wall (Garcia et al., 1987) and washed pellets have been shown to retain partial lignindegrading ability (Kurek and Odier, 1990). Although several works refer to the LiP of P.
chrysosporium as being the main decolorizing agent, a recent investigation of the
degradation of selected phthalocyanine dyes and their degradation products showed the
presence of Lac and MnP (Conneely et al., 2002) and the qualitative analysis of the
culture broths helped to propose a pathway for the catalytic process. The findings of
Kirby et al. (2000), 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 the
only 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, and
sustained repeated additions of individual dyes and dye mixtures in liquid cultures
(Swamy and Ramsay, 1999). Earlier results from the same group indicated the

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

177

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 carried
out without effluent (o) For decolorization of dye-containing effluent the cultures contained 10% (.) and 33%
(n) of raw wastewater in modified Kirk medium.

involvement of either a mycelial-bound LME or a H2O2-generating mechanism in the


cell 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 to
understand mechanisms. The proposed mechanism of 2,4,6-trinitrotoluene degradation
could also be involved in dye degradation (Stahl and Aust, 1993a,b; Van Aken and
Agathos, 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 fundamental question remains unanswered: Which are the points of primary attack for
decolorization and mineralization (e.g., the azo bond or the aromatic rings)? There is
still a lack of experimental evidence on toxic intermediate accumulation and a variability
of results among laboratories. Detoxification studies by Heinfling et al. (1997) have
shown that azo dye degradation by B. adusta results in nontoxic compounds. Different

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

specificities between peroxidase isoforms may help to explain variability in reported


results.

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, rotating
disks, etc., there is a dearth of analogous reports on the use of reactor systems
employing WRF for waste treatment. Towards the design of bioreactor systems for
decolorization, Zhang et al. (1999) used an alginate-immobilized basidiomycete,
producer of LiP, MnP and Lac, in several reactor configurations. The performance
of continuous packed bed, fedbatch fluidized bed and continuous fluidized bed
bioreactors in terms of decolorization rates and mycelium stability compared favorably
to previously reported continuous decolorization with a fixed film bioreactor using
immobilized P. chrysosporium (Yang and Yu, 1996). The adequate mass and oxygen
transfer in the fluidised-bed systems ensured reactor stability avoiding excessive
mycelial growth. It was suggested that the continuous fluidised-bed system, with
lower decolorization rates would be more suitable treating effluents at concentrations
that are inhibitory to the biomass whereas the fedbatch process would be better for
high but not inhibitory dye concentrations (Zhang et al., 1999).
The use of rotating biological contactors allows intermittent contact of the mycelium
with the effluent, thus avoiding overgrowth and the problems arising in packed-bed
reactors. It was shown that the efficiency of Coriolus (Trametes) versicolor (a well-known
decolorizer) in a rotating biodisk contactor varied depending on the biofilm thickness,
rotational speed and carbon source concentration (Kapdan and Kargi, 2002). However,
there is room for optimization of such systems.
Anaerobic decolorization of azo and other water-soluble dyes is possible via
oxidation reduction reaction with hydrogen, yielding methane and hydrogen sulphide
(Carliell et al., 1996). 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 projects (Robinson et al., 2001a).
Moreover, the toxic amines that are generated when azo and nitro compounds are reduced
(Banat et al., 1996; Beydilli et al., 1998), may create an additional pollution problem for
waterways.
The exposure of the reduced azo and nitro compounds to oxygen could cause
reappearance of coloration, however aromatic amines can be mineralized by nonspecific
enzymes through hydroxylation and ring-opening of the aromatic compounds under
activated sludge treatment. Coupled anaerobic aerobic degradation of dyes was tested by
ONeill et al. (2000), by means of HPLC-UV analyses. It was verified that toxic aminocontaining compounds were formed during the anaerobic stage of an Upflow Anaerobic
Sludge Blanket reactor for treatment of simulated textile effluent. During the succeeding
aerobic stage, some degradation of nitrogen-containing aromatic derivatives took place
with mineralization of organic nitrogen (ONeill et al., 2000). Heat-treatment liquor of

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

179

waste sludge was further treated in a fungal bioreactor coupled with ultrafiltration to
separate high molecular weight color components thus avoiding decolorization instability
caused by prolonged incubation (Fujita et al., 1994). Solid/liquid separations were easier
due to firm immobilization of fungal cells on polyurethane foam. This approach could be
also interesting for decolorization of synthetic dyes, but also for other persistent organic
pollutants.
Detailed studies on bioreactor performances are starting to emerge, seeking to extend the
capacity of WRF to decolorize dyes in continuous (Palma et al., 1999; Yang and Yu, 1996)
or sequencing batch mode (Borchert and Libra, 2001) over long periods of time without the
need for supplementation of new mycelium and, though a challenge, under nonsterile
conditions. Other promising technological developments with potential for enzymatic
treatment of effluents include the immobilization of laccase (Reyes et al., 1999) and the
coimmobilization of MnP with glucose oxidase for in situ H2O2 formation (Van Aken et al.,
2000b).

5. Perspectives
All three LME (MnP, LiP, Lac) are produced in multiple isoforms and encoded by
gene families with complex regulation. Nutrient levels, mediator compounds and
required metal ions (Mn2 + for MnP, Cu2 + for Lac) affect transcription of respective
genes. Judicious manipulation of the chemical environment may allow the production
of an adequate mixture of LME giving good decolorization without side products;
however, this approach is not optimal. Gene amplification and expression in appropriate
hosts could be promising for abundant production and affordable price of LME, as is
already the case with laccases used commercially in the pulp and paper industry.
Further potential benefits of genetically improved LME could be extended substrate
range, catalytic activity and stability for industrial application of LME.
Alternative systems, such as plants, could offer advantages over bacteria or fungi for
bioremediation purposes. Transgenic tobacco plants with LME genes from WRF are
currently proposed for the removal of hazardous chemicals from contaminated environments (Iimura et al., 2002) and could be applicable to the case of textile dyes.
As more insights are gained in understanding the chemical nature of recalcitrance,
enhanced transformation and, hopefully, complete mineralization of xenobiotic dyes could
be achieved by modular multistep processes, coupling reduction and oxidation reactions
by abiotic or biotic means (Rieger et al., 2002). This principle seems to be bearing fruit for
an increasing range of recalcitrant molecules.
Facing one of the most persistent groups of pollutants, organic dyes, modern
research communities are ultimately challenged to suggest alternative technologies
towards a better environment. A new generation of synthetic colorants for sustainability
would require the identification of structural analogs of natural compounds to be
introduced into new, benign by design products with both advanced technical
performance and biodegradability (Knackmuss, 2001). The combination of natural
building blocks (or biodegradable synthons) through hydrolyzable links (ester, amide,
acetal bonds) could ensure complete mineralization by relatively common microbial

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

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 towards
developing strategies for bioremediation. HPLC analyses for individual dyes are possible
in some cases, though this is labour intensive and probably not applicable for monitoring
complex transformations. Further studies should be conducted, using advanced analytical
techniques, to elucidate the catabolic processes involved in the degradation of distinct dye
groups by the LME of WRF. In the near future, the progress in the field of nanotechnology
could provide biochemical engineers powerful tools for studying cell surface and topology
to better understand the importance of membrane-bound oxidoreductases and their role in
growth-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 no
981/3870) 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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