J. Basic Microbiol.

27 (1987) 4, 179-184

(Department of Biosciences, M. D. University, Rohtak-124001, India' and Department of Biology,

Guru Nanak Dev University, Amritsar-143005, India*)

Decomposition of angiospermic wood sawdust and

laccase production by two Pleurotus species
D. S. ARORA~
and D. K. SANDHU~

(Received August 13, 1986; revision received December 29, 1986)

Out of different lignin preparations, phenolic compounds and sugars tested, indulin AT proved to
be the best substrate for laccase production in both Pleurotus ostreatus and P . sajor-caju. Sugars
supported good growth but repressed enzyme production. I n P. ostreatw the enzyme production
was maximum a t 25 "C, pH 5 and 0.1% indulin AT. Angiospermic wood saw dust suffered a total
weight loss of 6% accompanied by 14% lignin loss during 60 days incubation with P. ostreatus.

A lot of lignocellulosic residue gets accumulated every year in the form of leaf litter,
straw, wood shavings and sawdust etc. Lignin component is next to cellulose in orga-
nic carbon content. Despite its resistive nature it is decomposed by various microbes
to maintain balanced ecological set up and to provide energy-rich simple compounds.
Lignin component of lignocellulosics has to be removed for their efficient utilization
in paper industry or as feed for ruminants. Various workers have been involved in the
selective removal of lignin for biochemical pulping and to increase the digestibility of
agricultural residues for ruminants (KANESHIRO 1977, LINDENFELSER et al. 1979,
YANGet al. 1980, ZADRAZIL 1980, ERIESSON and VALLANDER 1981). Laccase has been
considered to be one of the important enzymes playing a key role in ligninolysis by
causing its demethylation (TROJANOWSKIand LEONOWICZ 1969, ANDER and ERIKSSON
1978). Various phenolic compounds have been used to enhance laccase production
in different fungi (FAHRAEUS 1954, FROEHNER and ERIKSSON 1974, ARORAand SANDHU
1984, SANDHU and ARORA1985). In the present study different physicochemical para-
meters have been standardized to optimize the enzyme production. Pleurotus ostreatus
was selected further to find out its rate of decomposing angiospermic wood sawdust
with special reference to lignin. An attempt has also been made to correlate its laccase
producing ability with lignin degradation.

Materials and methods

Microorganisms : The cultures of Pleurotus ostreutus (JACQ.) FR. and Pleurotus sujor-cuju (FR.)
SINGER (FRI983) were obtained from Punjab Agricultural University, Ludhiana and Forest Re-
search Institute, Dehradun, respectively. These were maintained on yeast glucose agar and
stored a t 4 "C.
Laccase production under different conditions: The two fungi were made to grow under a variable
set of physicochemical conditions so as t o optimize the laccase production. To find out its time
course the fungi were grown on lignin (indulin AT 1%)and malt extract (1%)media and processed
for enzyme estimation a t various time intervals (SANDHU and ARORA1984). I n order t o find out
the best suitable substrate for enzyme production by the two species the mineral solution was sup-
plemented with various lignins (indulin AT, polyfon, reax), phenolic compounds (gallic, tannic and
salicylic acids, orcinol, resorcinol) and sugars (glucose, maltose, lactose, sorbitol, sucrose) etc. as
described earlier (ARORAand SANDHU1984). Stimulatory effect, if any, of malt extract (0.2%)
was determined by supplementing the above media with it. Optimum temperature, pH and sub-
strate (indulin AT) concentration were standardized for P. ostreatus. Laccase estimations were
carried out using guaiacol as substrate and expressed in relative and specific activity units (SANDHU
and ARORA1985).
131
180 D. S. ARORAand D. K. SANDHU

Decomposition of wood by P. ostreatus: Pleurotus ostreatus was found to be better laccase pro-
ducer as compared to P. sujor-caju. Thus the former was grown on angiospermic wood sawdust to
determineits degradative ability in terms of total weight loss and lignin loss during two months of
decay (ARORAand SANDHU 1985).

Results and discussion

Laccase production under different conditions
The pattern of enzyme production in 1% lignin and malt extract media revealed better
laccase production in the latter medium in consonance with our earlier studies on
Daedalea flavida and Polyporus sanguineus with the maximum enzyme yields by P.
sanguineus (ARORAand SANDHU 1985, SANDHUand ARORA1985). However, lignin
medium was found to be the best for P. versicolor or P. hirsutus (SANDHUand ARORA
1984, ARORAand SANDHU1984). The enzyme yields obtained in these cases were cer-
tainly higher than that of Pleurotus species in malt extract medium. Pleurotus ostrea-
tus was unable to produce laccase in the lignin medium whereas Y . sajor-caju could do
so only to a low level (Fig. 1). Higher concentration of lignin may be responsible for
its inhibitory action as 0.1% was found t o be the best for enzyme production by P.
ostreatus in another set of experiment (Pig. 2C). Its inability t o produce enzyme in
lignin medium can also be attributed to its requirement for preadaptation t o such a
medium as observed in the case of Poria subacida and Polyporus abietinus (GOTTLIEB
1950). The maximum relative enzyme production in malt extract was recorded on 21st
and 7th day for P . ostreatus and P. sajor-caju, respectively, whereas maximum speci-
fic laccase activity was on 14th and 7th day (Fig. 1 A, B). The rate of enzyme produc-
tion did not find any correlation with biomass production a s the latter was higher in

0.6
0.5
-E.; ? 0.4
h

X r n
3
VlL:
5 0.3
0.2
21
+.
.- 0.1
.-c5
V
rd
Q)
UI
m
U
0
m
a 1.0 r x
I\
B

Fig. 1
Laccase productionby Pleurotus ostreatus (-)
and P. sajor-caju (---) on lignin (A) and
malt extract media ( x ). (A) relative activity,
(B) specific activity
7 14 21 28 35 42
Days
 &1:;
hecase production by Pleurotus species 181

0.50

- 0.25
h

E
\
f s
In

4
E
/ ‘JLx \
Y
20 30 40 F
Y

P)
In
.-
P)
3

a 3 4 5 6 7 8 9 D
-I

0.50

Fig. 2
0.25 Optimization of laccase production by Pleurotus
ostreatus. (A) temperature, (B) pH, (C) indulin AT
concentration
0.2 0.4 0.6 0 . 8
lndulin AT (*A)

P. sajor-caju. This is further supported by the observation that P. sangwineus, the

best enzyme producer on malt extract medium gave the lowest biomass (SANDKUand
ARORA1985).
Different lignin preparations, phenolic compounds and sugars gave a variable res-
ponse in the two fungi (Table 1) although the stimuluswas not as high as observed ear-
lier in other fungi (ARORAand SANDHU 1984, 1985, SANDHUand ARORA1984, 1985).
Supplementation of malt extract (0.2%)to these compounds enhanced the enzyme
production a step further which can be because of richness of malt extract to provide
complete pool of amino acids required for enzyme synthesis (FAHRAEUS 1954). Induc-
tion of laccase in the presence of lignin preparations and certain phenolic compounds
which was maximum in the case of indulin AT is in line with earlier observations
(TROJANOWSKI and LEONOWICZ 1969, ARORAand SANDHU1984, SANDHU and ARORA
1984, 1985). Of the different lignin preparations poor laccase activity in the presence
of reax can be attributed to its degree of sulfonation (4.7% as mentioned by its manu-
facturers, Westvaco Chemical Division, U.S.A.) as lignosulfonates have been shown to
be poorly utilized as compared to kraft lignin (ANDERand ERIKSSON 1978). Laccase
induced is suggested to detoxify the phenolic compounds (GRABBEet al. 1968, JOHANS-
SON and HAGERBY 1974) and cause demethylation of lignin, a key step for its degrada-
tion (TROXANOWSKI and LEONOWICZ 1969, KIRKand CHANG1975). Salicylic acid inhi-
bited enzyme as well as biomass production in both the fungi (Table 1). However, in
P. ostreatus orcinol and tannic acid also caused complete enzyme inhibition. The sugars
although good substrates for biomass production completely repressed the enzyme
activity in P.ostreatus and gave very low laccase yield in P.sajor-caju (Table 2 ) . The
enzyme repression in the presence of sugars may be due to catabolite repression (ANDER
and ERIKSSON 1975, WOOD1980). The optimum temperature, pH and substrate (indu-
182 D. S. ARORAand D. K. SANDHU

Table 1
Effect of different lignins and phenolic compounds on laccase production by Pleurotus ostreatus
(A) and P. sajor-caju (B) (Period of incubation - 10 days)

Substrate Laccase activity (CU/ml) Growth (mg/flask)*)

A B A B

Malt extract (M) 0.02 0.05 14 17

Indulin AT
+
M Indulin AT
0.01
0.27
0.10
0.42
+++ +++
Polyfon 0.03 0.03 4 9
+
M Polyfon 0.22 0.26 11 29
Reax 0.02 0.03 4.5 7
+
M Reax 0.21 0.15 10 26
Resorcinol 0.02 0.07 0 4
+
M Resorcinol 0.03 0.14 4 7
Gallic acid 0.00 0.01 3 7
+
M Gallic acid
Orcinol
0.04
-
0.12
0.02
12
-
23
3
M + Orcinol - 0.10 - 6
Tannic acid - 0.01 - 4
+
M Tannic acid - 0.04 - 7

*) + relative growth

Table 2
Effect of different sugars on laccase production by Pleurotus ostreatus (A) and P. sajor-caju (B)
(Period o f incubation - 7 days)

Substrate Laccase activity (CU/ml) Growth (mg/flask)

A B A B

Malt extract (M) 0.03 0.04 16 18

Glucose - 0.01 22.5 41
+
M Glucose 0.05 0.07 31 67
Lactose - 0.03 16 28
+
M Lactose 0.03
-
0.28 22.5 34
Sucrose 0.04 22.5 42
+
M Sucrose 0.02
-
0.20 31.5 44
Sorbitol 0.09 18.5 35
+
M Sorbital 0.02
-
0.25 32.5 37
Maltose 0.01 23 46
+
M Maltose 0.01 0.19 30 60

lin AT) concentration for laccase production by P. ostreatus were recorded to be 25 "C,
5.0 and O.lyo,respectively.

Degradation of wood sawdust b y P. ostreatus

Wood sawdust was degraded at an almost uniform rate to cause a total weight loss of
2.7% during first 30 days which reached upto 6% in the next 30 days of decay. A lig-
nin loss of 8.5% and 14.4% was recorded on 30 and 60 days of incubation, respectively.
Comparatively more selective removal of lignin observed during early stages of degra-
dation is in agreement with previous studies on P. ostreatus and other white-rot fungi
where lignin loss occurred prior to that of cellulose (LINDENFELSER et al. 1979, ANDER
and ERIKSSON 1978, ARORAand SANDHU 1984). The initial lignin degradation might
have been favoured by the malt extract by providing precursors for enzyme synthesis
and acting as a cosubstrate to yield necessary energy for ligninolysis (ANDERand ERIKS-
Laccase production by Pleurotus species 183

SON 1975, ERIKSSON et al. 1980). Higher laccase production (0.28 CU/ml on 30th day)
in earIy stages may be responsible €or concomitant higher lignin loss thus supporting
the involvement of laccase in ligninolysis (ANDERand ERIKSSON 1976). Polyporus ver-
sicolor, a better enzyme producer, also showed better lignin decomposition (ARORAand
SANDHU 1986) which corroborates the present observations.

Acknowledgements
The authors are thankful to Punjab Agricultural University, Ludhiana ; Forest Research Institute,
Dehradun and Westvaco Chemical Division, USA for providing Pleurotua oatreatus, P. sajor-caju
and lignin samples, respectively. DSA extends his thanks to University Grants Commission, New
Delhi, India for providing financial assistance.

References
ANDER, P. and ERIKSSON, K. E., 1975. Influence of carbohydrates on lignin degradation by the
white-rot fungus Sporotrichum pulverulentum. Sven. Papperstidn., 18, 643-652.
ANDER,P. and ERIKSSON, K. E., 1976. The importance of phenol oxidase activity in lignin degra-
dation by the white-rot fungus Sporotrichum pulverulentum. Arch. Microbiol., 109, 1-8.
ANDER,P. and ERIKSSON,K. E., 1978. Lignin degradation and utilization by microorganisms.
Prog. Ind. Microbiol., 14, 1-58.
ARORA,D. S. and SANDHU, D. K., 1984. Laccase production and wood degradation by Trametes
hirsuta. Folia Microbiol., 29, 310-315.
ARORA,D. S. and SANDHU, D. K., 1985. Laccase production and wood degradation by a white-rot
fungus Daedalea flavida. Enzyme Microb. Technol., 7, 405-408.
ARORA, D. S. and SANDHU, D. K., 1986. Degradation of lignocellulosic residues by Polyporus ver-
sicolor and the effect of moisture contents and phenolic compounds. Acta Biotechnol., 6,251 to
255.
ERIKSSON, K. E. and VALLANDER, L., 1981. Biochemical pulping. I n : Lignin Biodegradation:
Microbiology, Chemistry and Potential Applications (T. K. KIRK, T. HIGUCHIand H. CHANG,
Eds.), Vol. 2, pp. 213-224. Boca Raton, Florida.
ERIKSSON, K. E., GRUNNEWALD, A. and VALLANDER, L., 1980. Studies of growth conditions in
wood for three white-rotfungiand their cellulase-lessmutants. Biotechnol. Bioeng., 22,363-376.
FAHRAEUS, G., 1954. Further studies in the formation of laccase by Polyporus versicolor. Physiol.
Plant., 7, 704-712.
FROEHNER, S. C. and ERIKSSON, K. E., 1974. Induction of Neurospora crassa laccase with protein
synthesis inhibitors. J. Bacteriol., 120, 450-457.
GOTTLIEB, S., DAY,W. C. and PELCZAR, M. J., 1950. The biological degradation of lignin. 11. The
adaptation of white-rot fungi to growth on lignin media. Phytopathol., 40,926-935.
GRABBE, K., KOENIG,R. und HAIDER,K., 1968. Die Bildung der Phenoloxydase und die Stoff-
wechselbeeinflussung durch Phenole bei Polystictus versicolor. Arch. Mikrobiol., 63, 133 -153.
JOHANSON, M. and HAGERBY, E., 1974. Influence of growth conditions, metabolic inhibitors, and
phenolic compounds on the ATP pool of Fomes annosus. Physiol. Plant., 32,23-32.
KANESHIRO, T., 1977. Lignocellulosic agricultural wastes degraded by Pleurotus ostreatus. Dev.
Ind. Microbiol., 18, 591 -601.
KIRK,T. K. and CHANG,H. M., 1975. Decomposition of lignin by white-rot fungi 11.Characteriza-
tion of heavily degraded lignins from decayed spruce. Holzforschung, 29,56-64.
LINDENFELSER, L. A., DETROY,R. W., RAMSTACK, J. M. and WORDEN,K. A., 1979. Biological
modification of the lignin and cellulose components of wheat straw by Pleurotus ostreatus. Dev.
Ind. Microbiol., 20, 541-551.
SANDHU, D. K. and ARORA, D. S., 1984. Laccase production by Polyporus versicolor on different
substrates. Acta Biotechnol., 4, 49-57.
SANDHU, D. K. and ARORA,D. S., 1985. Laccase production by Polyporus sanguineus under diffe-
rent nutritional and environmental conditions. Experientia 41,355 -356.
TROJANOWSKI, J. and LOENOWICZ, A., 1969. The biodeterioration of lignin by fungi. Microbios., 8,
247-251.
184 D. S. ARORA and D. K. SANDHU

WOOD,D. A., 1980. Production, purification and properties of extracellular laccase of Agaricue
biepOru8. J. Gen. Microbiol., 117,327-338.
YANQ,H. H., EFFLAND, M. J. and KIRK,T. K., 1980. Factors influencing fungal degradation of
lignin in a representative lignocellulosic thermomechanical pulp. Biotechnol. Bioeng., 22, 65 t o
78.
ZADRAZIL,
F., lB80. Conversion of different plant waste into feed by basidiomycetes. Eur. J. Appl.
Microbiol. Biotechnol., 9, 243-248.

Mailing address: Dr. D. K. SANDHU,

Department of Biology, Guru Nanak Dev University, Amrit-
sar-143005. India