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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
162
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
163
Table 2
Classes of organic dyes: structures of representative colorants
164
their lack of substrate specificity, are also capable of degrading a wide range of
xenobiotics.
165
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
166
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
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
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
168
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)
Li et al., 1999
169
Reaction 2
Reaction 3
Reaction 4
Reaction 5
Reaction 6
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
170
(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).
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)
Ceriporia
metamorphosa
Daedalea flavida
Daedaleopsis
confragosa
Dichomitus squalens
Geotrichum
candidum
Irpex lacteus
Lentinus tigrinus
ND
Mycoacia nothofagi
Phanerochaete
chrysosporium
|LiP|MnP (Kaal
et al., 1995)
(Lac)|LiP|MnP
(Cameron et al., 2000)
172
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
Piptoporus betulinus
Pleurotus eryngii
Pleurotus ostreatus
Pleurotus sajor-caju
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)
173
Table 5 (continued)
Organism
LMEa (Reference)
Dyeb (Reference)
Polyporus ciliatus
Polyporus
sanguineus
Pycnoporus
sanguineus
Stereum hirsutum
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
174
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
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).
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 (- - - - -).
176
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
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)
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
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.
178
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
180
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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