Bioresource Technology 77 (2001) 89±91

Short communication

E€ects of various media and supplements on laccase production by

some white rot fungi
Daljit Singh Arora *, Paramjit Kaur Gill
Microbial Technology Laboratory, Department of Microbiology, Guru Nanak Dev University, Amritsar, 143005 Punjab, India
Received 22 May 2000; received in revised form 10 August 2000; accepted 22 August 2000

Abstract
White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese
peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The
present paper reports its production by some white rot fungi; Daedalea ¯avida, Phlebia brevispora, Phlebia radiata and Polyporus
sanguineus under di€erent nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the
best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added
to di€erent media. Ó 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Laccase; Lignin; White rot fungi; Inducers

1. Introduction continuation of the earlier work and reports laccase

production by some more white rot fungi; Daedalea
Laccase, lignin peroxidase and manganese peroxidase ¯avida, Phlebia brevispora, P. radiata and Polyporus
are among the major enzymes of white rot fungi in- sanguineus.
volved in lignin degradation. Ligninolytic potential of
laccase has been exploited in biopulping and bioble-
aching (Call, 1994), improving the digestibility of
2. Methods
lignocellulosics (Martinez et al., 1994) and removal of
phenolic compounds from e‚uents, must, wine and fruit
2.1. Microorganisms
juices (Cantarelli et al., 1989; Giovanelli and Ravasini,
1993).
Four white rot fungi; D. ¯avida (MTCC 145),
Laccases (benzenediol: oxygen oxidoreductase;
P. brevispora (HHB-7030-Sp), P. radiata (MJL-1198-Sp)
EC1.10.3.2) of some white rot fungi have been puri®ed
and Polyporus sanguineus (MTCC 137), were selected
and characterized biochemically (Fahraeus and Rein-
for the present study. D. ¯avida and P. sanguineus were
hammer, 1967; Wood, 1980; Oda et al., 1991). However,
procured from the Institute of Microbial Technology,
not many systematic and comparative studies are
Chandigarh. Phlebia spp. were received as a gift from
available on quantitative laccase production. The im-
Dr. C.R. Bergmann, F.P.L., Madison, Wisconsin. The
portance of laccase in various biotechnological areas
cultures were maintained on glucose yeast extract agar
underlines the need for expanding the spectrum of lac-
(YGA) slants and stored at 4 C.
case-producing organisms and enhancing the potential
of their laccase-producing ability. In our earlier study
laccase production by the much less-studied Dichomitus 2.2. Laccase production in di€erent basal media and e€ect
squalens, Phlebia fascicularia and P. ¯oridensis was re- of di€erent supplements
ported (Arora and Gill, 2000). The present study is a
Laccase production was studied in three basal media,
*
Corresponding author. Tel.: +91-258802-09; fax: + 91-183-258819.
mineral salts broth (MSB), malt extract broth (MEB)
E-mail addresses: daljit_arora@goplay.com, jcse.gndu@yahoo.com and mineral salts malt extract broth (MSB-MEB).
(D.S. Arora). Various supplements were added to di€erent basal

0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 1 4 - 0
90 D.S. Arora, P.K. Gill / Bioresource Technology 77 (2001) 89±91

media to study their e€ect on laccase production as
described previously (Arora and Gill, 2000).
3. Results and discussion
3.1. Laccase production in di€erent basal media
Better enzyme production was obtained in MEB or
MSB-MEB in comparison to MSB alone. Malt extract is
rich in the aromatic amino acids tryptophan and tyro-
sine. Eggert et al. (1996b) identi®ed a tryptophan-de-
rived metabolite (3-hydroxy-2-aminobenzoate) acting as
a mediator in laccase-catalyzed oxidation reactions in
the white rot fungus Pycnoporus cinnabarinus. More-
over, laccase is known to react with 4-hydroxyindole, a
tryptophan derivative (Cai et al., 1993; Eggert et al.,
1996a). Collins et al. (1997) reported a large increase in
lignin peroxidase production on adding tryptophan to
the cultures of Trametes versicolor, Phanerochaete
chrysosporium and Chrysosporium lignorum, thus indi-
cating that tryptophan, as provided by malt extract,
might be responsible for better enzyme yields. In the
present study P. radiata gave maximum laccase pro-
duction (6.18 cu/ml) in MSB-MEB (Table 1) which was
the best nutritive medium for the high laccase producer,
P. fascicularia as evident from our previous study
(Arora and Gill, 2000).
3.2. E€ect of di€erent nutritional supplements
The e€ect of veratryl alcohol on laccase production
varied with the organism and also with the basal media
used (Table 1) which is in consonance with earlier
studies where di€erent white rot fungi responded dif-
ferentially towards laccase enhancement by low molec-
ular weight aromatic compounds (D'souza et al., 1999).
Veratryl alcohol on supplementation to MSB enhanced
laccase production to more than two hundred-fold in
P. radiata. On the other hand, in D. ¯avida, P. brevispora
and P. sanguineus 2- to 6-fold stimulation in laccase
production was observed on adding veratryl alcohol to
MEB. The inductive e€ect of veratryl alcohol on laccase
production by Dichomitus squalens, Ganoderma lucidum,
P. fascicularia, P. ¯oridensis, P. cinnabarinus and
T. versicolor has been reported in the earlier studies
(Arora and Gill, 2000; Bollag and Leonowicz, 1984;
Schlosser et al., 1997).
Guaiacol supplementation to all the media enhanced
laccase production from 2- to 232-fold in di€erent fungi.
Similar to veratryl alcohol, guaiacol also gave maximum
stimulatory e€ect in P. radiata (232-fold) followed by
D. ¯avida (20-fold) on its supplementation to MSB and
MEB, respectively (Table 1). Comparison of the data
presented here and earlier studies (Arora and Gill,
2000), revealed Phlebia spp. to be more sensitive to the
inductive e€ect of guaiacol as compared to other white

Table 1
Laccase production by di€erent white rot fungi in di€erent media/supplements
Media Laccase activity (cu/ml)
D. ¯avida P. brevispora P. radiata P. sanguineus
MSBa 0.060 0.350 0.045 0.130
MEBb 0.050 1.370 0.415 0.672
MSB ) MEBc 0.660 4.045 6.180 0.502
MSB + VAd 0.150 0.165 9.470 0.055
MEB + VA 0.332 2.835 0.045 1.745
MSB ) MEB + VA 0.640 4.175 11.185 0.235
MSB + Ge 0.217 0.605 10.475 0.647
MEB + G 1.042 5.695 2.490 4.765
MSB ) MEB + G 1.24 5.700 11.590 1.617
MSB + indulin AT 4.52 2.765 7.893 1.367
MSB + polyfon 6.05 3.170 6.090 3.125
MSB + reax 2.74 2.350 7.010 2.800
MSB + lignosulphonate 0.807 6.565 6.770 1.042
MSB + WSf 9.25 8.90 9.120 2.740
MEB + WS 8.49 5.885 5.320 8.330
MSB + RSg 9.04 5.290 8.795 3.057
MEB + RS 3.18 6.605 1.435 4.50
MSB + SBh 5.62 8.130 10.03 1.725
MEB + SB 3.30 8.560 8.90 8.99
a
Mineral salts broth.
b
Malt extract broth.
c
Mineral salts-malt extract broth.
d
Veratryl alcohol.
e
Guaiacol.
f
Wheat straw.
g
Rice straw.
h
Sugarcane bagasse.
 D.S. Arora, P.K. Gill / Bioresource Technology 77 (2001) 89±91
rot fungi. Such inductive e€ect of guaiacol has earlier
been reported in Stereum hirsutum, Marasmius gramin-
ium (Fahraeus et al., 1958) and Coprinus friesii (Heinz-
kill, 1995) though laccase production was suppressed by
guaiacol in Pleurotus ¯orida and P. cinnabarinus
(Dhaliwal et al., 1992; Eggert et al., 1996a). This might
be due to the higher concentration of guaiacol used in
these studies.
Phlebia spp. are more responsive to the supplemen-
tation of lignin preparations, with indulin AT being the
best stimulator for P. radiata. Di€erent lignin prepara-
tions have been reported earlier to act as inducers for
laccase production, indulin AT for Lentinula edodes,
lignin and lignosulphonate for P. ¯orida and P. cinna-
barinus, respectively (Dhaliwal et al., 1992; Eggert et al.,
1996a; Savoie et al., 1998). Some degradation product/s
of lignin might be acting as inducer for laccase.
Phlebia radiata and D. ¯avida were more responsive
to the inductive e€ect of various agricultural residues.
Laccase production by P. radiata was enhanced 222
times on addition of sugarcane bagasse to MSB whereas
in D. ¯avida the enhancement was about 170 times on
supplementing wheat straw to MEB. Sugarcane bagasse
has been reported to be a better substrate for laccase
production by Flavodon ¯avus also (Raghukumar et al.,
1999).
In conclusion, a look at the present and previous
studies clearly reveals that P. radiata, P. fascicularia and
P. ¯oridensis are the best white rot fungi for laccase
production. D. ¯avida is another promising organism in
this area. The enzyme production ability of these org-
anisms can be enhanced several fold on supplementing
the basal media with various natural or chemically
synthesized materials. Moreover, their enzyme systems
seemed to be much more responsive to the inductive
e€ect of various supplements, of which veratryl alcohol,
guaiacol, indulin AT, sugarcane bagasse and wheat
straw were excellent laccase inducers.
Acknowledgements
We are thankful to Dr. C.R. Bergmann, Forest
Products Laboratory, Madison, Wisconsin for provid-
ing us the cultures of Phlebia spp.
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