Journal of Scientific & Industrial Research
1068
Vol. 68, December 2009, pp. 1068-1074
J SCI IND RES VOL 68 DECEMBER 2009
Influence of pH, temperature and cultural media on decolorization of
synthetic dyes through spent substrate of different mushrooms
O.P. Ahlawat* and Rajender Singh
Directorate on Mushroom Research (ICAR), Chambaghat, Solan 173 213, India
Received 30 July 2009; accepted 23 October 2009
Spent substrate of Agaricus bisporus, Pleurotus sajor-caju and Lentinula edodes was evaluated for decolorization of 4
different dyes under different cultural conditions (media, incubation temperature and medium pH). Potato dextrose broth media
supported highest decolorization (90% of Rhodamine B and Methyl Violet 2B, 93% of Chicago Sky Blue 6B and 88% of
Quinaldine Red) after 3 days of incubation with P. sajor-caju spent substrate. Optimum pH requirements were 7.0 and 10.0 for
Rhodamine B and Methyl Violet 2B with spent substrate of L. edodes, while 4.0 and 7.0 with A. bisporus and P. sajor-caju.
However, for Chicago Sky Blue 6B and Quinaldine Red, it was 4.0 and 7.0 for spent substrates of all 3 mushrooms. Spent
substrate of A. bisporus supported highest decolorization of Rhodamine B (93-95%) and Methyl Violet 2B (91-100%), followed
by L. edodes + P. sajor-caju. Temperatures of 25 and 35oC were most effective for decolorization of Rhodamine B (90%) and
Methyl Violet 2B (97%) with spent substrate of A. bisporus alone. In case of Chicago Sky Blue 6B and Quinaldine Red,
temperature requirement for decolorization was same but with spent substrate of L. edodes, P. sajor-caju and L. edodes + A.
bisporus for Chicago Sky Blue 6B, and A. bisporus, P. sajor-caju and L.edodes + A. bisporus for Quinaldine Red.
Keywords: Agaricus bisporus, Dye decolorization, Lentinula edodes, Pleurotus sajor-caju, Spent mushroom substrate
Introduction
During industrial processing, used dyestuffs
(up to 40%) are released into processed water, producing
highly colored wastewater that affects aesthetics,
transparency and gas solubility in water bodies1. Other
main factors are mutagenic, carcinogenic and toxic
effect of some of these dyes2, high volume and effluent
composition3 and their resistance towards conventional
methods of wastewater treatment2. Some physicochemical methods, available for reduction of several
dyes, require highly expensive catalysts and reagents4.
Biotechnological approaches4 mainly white rot fungi5
have shown effectiveness in treatment of dyes in an
eco-efficient manner.
Spent mushroom substrate (SMS), released after
cultivation of different mushrooms, contains residual
mushroom fungal biomass along with a rich population
of heterotrophic fungi and bacteria, which can act as an
inexpensive source of phenoloxidases. SMS also has
ability to chemically adsorb organic and inorganic
pollutants, while diverse category of microbes it harbors
have capability of biologically breaking down of organic
*Author for correspondence;
Tel: +91-1792-230767; Fax: +91-1792-231207
E-mail: [email protected]
xenobiotic compounds present in soil and water6. Role of
extracellular ligninolytic enzymes and microbes from SMS
of different mushrooms 7 , and mushroom mycelia,
especially of Pleurotus florida, P. ostreatus, P.
flabellatus and P. sajor-caju, have been evaluated for
use in dye decolorization activities3,8,9.
This study presents role of pH, temperature and type
of growth medium, if any, in decolorization of synthetic
dyes using spent substrate of A. bisporus, P. sajor-caju
and L. edodes.
Materials and Methods
Substrates and Chemicals
Four dyes [Rhodamine B10 (C28H31N2O3Cl), Methyl
Violet 2B11 (C25H30ClN3), Quinaldine Red12 (C21H23IN2)
and Chicago Sky Blue 6B13 (C34H24N6Na4O16S4)] were
procured from Sigma-Aldrich (Fig. 1). Hydrochloric acid
(HCl) and Sodium hydroxide (NaOH) used for pH
adjustment were from Merck. Samples of spent substrates
of button mushroom (Agaricus bisporus strain, A-15),
shiitake mushroom (Lentinula edodes strain, OE-142) and
oyster mushroom (Pleurotus sajor-caju strain, PL-1140)
were collected from Directorate of Mushroom Research,
Solan, HP, India.
AHLAWAT & SINGH: SPENT SUBSTRATE OF MUSHROOMS FOR DYE DECOLORIZATION
1069
Fig 1— Chemical structures of different dyes
Cultural Media Studies
pH
Three different growing media [malt extract broth
(MEB), potato dextrose broth (PDB) and plain water
(PW)], procured from Hi-Media Labs, were used for
studying effect of media composition on decolorization
of 4 dyes by using spent substrate of oyster mushroom
(P. sajor-caju), which is considered as potential dye
decolorizer among basidiomycetous fungi. Media (100
ml each) of MEB (malt extract, 25 g; distilled water,
1000 ml; pH, 7.2), PDB (potato dextrose, 24 g; distilled
water, 1000 ml; pH, 7.2) and PW was poured in
Erlenmeyer flasks (250 ml) and sterilized at 15 psi for 20
min. The 0.1 ml of stock solution (1 g/10 ml water) of
each dye was added in pre-sterilized growing media flasks
to make up 100 ppm concentration of each dye, followed
by addition of 1 g spent substrate of P. sajor-caju in
each flask using sterilized forcep. Experiment was carried
out in triplicate and all flasks were incubated at 25 ± 1oC
for next 5 days. Decolorization, if any, was recorded by
recording decrease in optical density at λmax from 0 day
up to 5 days of incubation by keeping flasks devoid of
SMS as control treatment.
Three different pH (4.0, 7.0 and 10.0) were used for
studying effect of pH on decolorization of four dyes with
spent substrates of white button (A. bisporus), shiitake
(L. edodes) and oyster (P. sajor-caju) mushrooms. PDB
was prepared by adjusting pH with 0.1N solution of HCl
or NaOH. Prepared broth was distributed in 250 ml
Erlenmeyer flasks @ 100 ml each and sterilized at 15 psi
for 20 min. The 0.1 ml stock solution (1 g/10 ml) of each
dye was added in 100 ml sterilized broth in flasks to give
a final concentration of 100 ppm and 1.0 g of spent
substrate from all 3 mushrooms was added separately as
well as in different permutations and combinations using
sterilized forceps. Flasks devoid of SMS but with different
dyes were kept as control treatments. Three replications
were kept for each treatment and all flasks including control
were incubated at 25 ± 1oC for next 5 days. Before
recording optical density, 3 ml sample from each flask
was withdrawn and centrifuged (Sigma centrifuge) at
10,000 rpm for 10 min and clear supernatant was used
for recording optical density. Decolorization, if any, was
recorded by recording decrease in optical density at λmax
1070
J SCI IND RES VOL 68 DECEMBER 2009
120
Decolorization, %
100
80
MEB
60
PD B
40
PW
20
0
RB
MV 2B
CSB 6B
QR
Media
Fig. 2— Effect of cultural media in decolorization of different dyes using spent substrate of
P. sajor-caju
using UV-Visible double beam Spectrophotometer
(Unico-3802) starting from 0 day to 5 days of incubation.
Decolorization extent (%) = [100 x (OD1-ODt)]/OD1
…(1)
Temperature
where OD1 is initial absorbance at 0 day, OD t is
absorbance after incubation for different periods under
different experimental conditions, t is incubation time.
Three different temperatures (15, 25 and 35oC) were
used for studying effect of different temperatures on
decolorization of four dyes using SMS of oyster
(P. sajor-caju), white button (A. bisporus) and shiitake
(L. edodes) mushrooms. Protocol for media preparation
and setting up of experiment was same as was for pH
studies, excepting medium pH, which was kept 7.2.
Experiment was carried out in triplicate and all flasks
including control were incubated at 3 different
temperatures (15, 25 and 35oC) for next 5 days. Before
recording optical density, sample (3 ml) from each flask
was withdrawn and centrifuged at 10,000 rpm for 10 min
and clear supernatant was used for recording
decolorization, if any, by recording decrease in optical
density at λmax from 0 day to 5 days of incubation. Flasks
devoid of SMS were kept as control treatment.
Measurement of Decolorization Extent
Sample (3 ml) collected each time from each
replication and centrifuged at 10000 rpm for 10 min was
used for measuring decolorization extent by measuring
absorbance of supernatant at specific λmax for each dye
by using UV-Visible double beam Spectrophotometer
(Unico-3802). Decolorization extent was calculated as
Results and Discussion
Role of Medium
Highest decolorization of Methyl Violet 2B (63%)
and Quinaldine Red (76%) after 24 h of incubation was
observed in PW. However, highest decolorization of
Chicago Sky Blue 6B (46%) and Rhodamine B (17%)
was in PDB. Although decolorization of dyes increased
with time up to 4 days of incubation, however, major
enhancement (Rhodamine B and Methyl Violet 2B, 90%;
Chicago Sky Blue 6B, 93%; and Quinaldine Red, 88%)
was recorded up to 3 days of incubation. Preferences
towards cultural media remained same throughout the 5
days of incubation (Fig. 2). In present study, difference
in decolorization of 4 dyes on using different cultural
media is attributed to variations in nutritional composition
of three media; as this variation also contributes towards
variation in their ability to sustain growth and multiplication
of different types of microorganisms contributed by P.
sajor-caju spent substrate. In some cases, higher
decolorization in PW can be ascribed to residual
extracellular ligninolytic enzymes and microbes
AHLAWAT & SINGH: SPENT SUBSTRATE OF MUSHROOMS FOR DYE DECOLORIZATION
pH 4.0
pH 7.0
pH 10
pH 4.0
pH 7.0
1071
pH 10
Decolorization, %
120
100
80
60
40
20
0
RB
MV 2B
CSB 6B
QR
A. b isporus L. edodes A. b isporus L. edodes A. b isporus L. edodes A. b isporus L. edodes
Dye/SMS
Fig 3— Effect of pH of the cultural medium on decolorization of different dyes with spent substrate of
A.bisporus and L.edodes
Table 1— Effect of pH of growing medium on decolorization of Rhodamine B and Methyl Violet 2B with spent substrates of different
mushrooms
Spent substrate
Day 1
L. edodes
A. bisporus
P. sajor-caju
L. edodes + A. bisporus
L. edodes + P. sajor-caju
A. bisporus + P. sajor-caju
CD
0.05
4.0
42
66
28
1
77
2
7.0
51
73
37
1
83
0
Dye decolorization (%) at different pH at different interval (days)
Rhodamine B
Methyl Violet 2B
Day 3
Day 1
Day 3
10.0
57
58
47
0
53
0
4.0
83
93
57
67
91
87
7.0
87
95
75
64
93
89
10.0
92
93
76
43
84
85
4.0
61
87
94
0
85
59
7.0
71
65
72
0
82
74
10.0
65
0
46
16
71
70
4.0
75
100
98
91
94
98
7.0
98
98
92
95
96
94
10.0
97
91
84
74
85
91
; pH, 8.29; days, 9.59; dyes, 7.08; spent substrates, 6.7; spent substrates × dyes × pH × days, 6.17
contributed by SMS, and least chances of enzymatic
inhibition compared with other cultural media. Role of C
and N in dye decolorization has been reported23-24 and
almost in all cases addition of glucose is reported to
enhance rate of dye decolourization.
Effect of Medium pH
In decolorization of Rhodamine B and Methyl Violet
2B, SMS of different mushrooms was effective in
decolorization at different pH values; SMS of L. edodes
was most effective at pH 7.0 and 10.0, while of
A. bisporus and P. sajor-caju at 4.0 and 7.0. Highest
decolorization of Rhodamine B (93-95%) and Methyl
Violet 2B (91-100%) was with A. bisporus SMS
(Fig. 3), followed by L.edodes + P. sajor-caju after 3
days of incubation (Table 1). In case of Chicago Sky
Blue 6B and Quinaldine Red, decolorization was almost
similar at pH 4.0 and 7.0 on using SMS of all three
mushrooms and it was higher in comparison to
decolorization at pH 10.0 (Fig. 3). Combined inoculums
of SMS of different mushrooms did not show much
superiority over SMS from individual mushroom, with an
exception of L. edodes + P. sajor-caju SMS for Chicago
Sky Blue 6B and Quinaldine Red (Table 2). Highest
decolorization of Chicago Sky Blue 6B (95-99%) and
Quinaldine Red (98-100%) after 3 days on incubation
was with SMS of L. edodes and L. edodes + P. sajorcaju, respectively. In all dyes, decolorization increased
only marginally from 3 days up to 5 days of incubation.
Studies 14-16 indicated an optimum pH for dye
decolorization through a specific microorganism. A vide
variation in pH requirements for decolorization of different
1072
J SCI IND RES VOL 68 DECEMBER 2009
Table 2— Effect of pH of growing medium on decolorization of Chicago Sky Blue 6B and Quinaldine Red with spent substrates of
different mushrooms
Spent substrate
Dye decolorization (%) at different pH at different interval (days)
Chicago Sky Blue 6B
Quinaldine Red
Day 1
L. edodes
A. bisporus
P. sajor-caju
L. edodes + A. bisporus
L. edodes + P. sajor-caju
A. bisporus + P. sajor-caju
CD
0.05
4.0
20
58
27
49
44
68
Day 3
7.0
38
46
35
61
71
71
10.0
5
11
6
0
13
22
4.0
95
84
95
67
94
88
Day 1
7.0
99
79
92
71
93
88
10.0
82
41
61
7
38
58
4.0
82
85
86
6
99
98
7.0
75
90
86
8
88
78
Day 3
10.0
71
36
77
3
85
74
4.0
86
90
90
94
100
98
7.0
87
88
88
96
98
96
10.0
80
80
83
72
93
89
; pH, 9.17; days, 8.32; dyes, 8.04; spent substrates, 5.8; spent substrates × dyes × pH × days, 6.05
Table 3— Effect of temperature of incubation on decolorization of Rhodamine B and Methyl Violet 2B with spent substrates of
different mushrooms
Spent substrate
Dye decolorization (%) at different temperatures at different interval (days)
Rhodamine B
Methyl Violet 2B
Day 1
L. edodes
A. bisporus
P. sajor-caju
L. edodes + A. bisporus
L. edodes + P. sajor-caju
A. bisporus + P. sajor-caju
CD
15
2
0
2
0
0
0
25
4
33
16
0
10
1
Day 3
35
43
83
6
70
49
56
15
50
81
50
86
79
72
25
52
85
46
91
90
85
Day 1
35
73
90
51
87
84
88
15
37
19
64
1
46
27
25
63
44
80
13
59
67
Day 3
35
77
79
83
82
66
89
15
79
87
93
69
82
78
25
82
97
93
84
84
85
35
93
97
88
89
79
84
; temp., 7.47; days, 9.98; dyes, 7.22; spent substrates, 2.698; spent substrates × dyes × pH × days, 6.42
0.05
dyes through different microorganisms has been cited
and it varied from 10 in case of Clostridium bifermentans
SL186 for Reactive dyes15, 7.0 in Citrobacter sp. CK3
for Reactive Red16 and 5.0 in Aspergillus niger SA1 for
Acid Red 15114. pH of growing medium has not only
been reported to support growth of specific
microorganisms but it has also been reported to play role
in activity of extracellular ligninolytic enzymes having
direct role in dye decolourization process3,17, hence whole
decolorization process has been found to be affected by
pH of cultural medium. In present study, dyes have been
recorded to have different pH optima for decolorization
through SMS of different mushrooms, as spent substrates
from three different mushrooms vary in their physicochemical properties; hence also in inhabiting microbial
population18. Although studies3,9,19 highlighted role of
white rot fungi including Pleurotus spp. in dyes
decolorization, but none highlighted role of spent substrate
of different mushrooms in decolorization of different dyes,
excepting some preliminary studies, wherein SMS of A.
bisporus performed better than SMS of Pleurotus spp.7.
Effect of Temperature
At an initial stage (after 1 day), decolorization of
Rhodamine B and Methyl Violet 2B was higher at 35oC
than at other two temperatures. However, after 3 days
of incubation, there was marginal difference in levels of
decolorization at 25 and 35oC. In some treatments
(P. sajor-caju SMS and P. sajor-caju + L.edodes SMS),
decolorization recorded at 15oC was also comparable to
that of other temperatures (Table 3). Combined SMS
treatments did not show any superiority over individualized
SMS treatments; as decolorization of Rhodamine B was
at par in A.bisporus alone (90%) and A.bisporus +
L edodes (91%) SMS treatments, while in case of Methyl
Violet 2B, it was highest in A. bisporus (97%) and
P. sajor-caju (93%) SMS treatments (Fig 4). In case of
Chicago Sky Blue 6B and Quinaldine Red, almost similar
trend was recorded at initial stage of decolorization.
However, at later stage, incubation temperatures of 25
and 35oC were equally effective in decolorization of 2
dyes, particularly with SMS of L. edodes, P. sajor-caju
and L. edodes + A. bisporus for Chicago Sky Blue 6B,
and A. bisporus, P. sajor-caju and L. edodes + A.
AHLAWAT & SINGH: SPENT SUBSTRATE OF MUSHROOMS FOR DYE DECOLORIZATION
15º C
120
25º C
35º C
15º C
25º C
1073
35º C
Decolorization, %
100
80
60
40
20
0
RB
RB
A. b isporus
MV 2B
MV
2B
L. edodes
A. b isporus
CSB 6B
CS
B 6B
L. edodes
A. b isporus
L. edodes
QR
QR
A. b isporus
L. edodes
Dye/SMS
Fig 4— Effect of temperature of incubation on decolorization of different dyes with spent substrate of
A. bisporus and L. edodes
Table 4—Effect of temperature of incubation on decolorization of Chicago Sky Blue 6B and Quinaldine Red with spent substrates of
different mushrooms
Spent substrate
Dye decolorization (%) at different temperatures at different interval (days)
Chicago Sky Blue 6B
Quinaldine Red
Day 1
L. edodes
A. bisporus
P. sajor-caju
L. edodes + A. bisporus
L. edodes + P. sajor-caju
A. bisporus + P. sajor-caju
CD
0.05
15
4
6
22
8
34
21
25
26
50
79
49
47
69
Day 3
35
55
65
94
77
65
71
15
54
87
73
50
68
70
25
94
86
98
86
79
79
Day 1
35
97
98
98
87
88
80
15
40
3
66
16
55
25
25
45
91
84
54
65
70
Day 3
35
67
93
81
84
70
79
15
64
91
80
58
62
70
25
81
94
86
84
79
80
35
88
95
82
86
74
84
; Temp., 9.01; days, 8.46; dyes, 6.29; spent substrates, 5.337; spent substrates × dyes × pH × days, 4.99
bisporus for Quinaldine Red (Table 4). Here again,
individualized SMS treatments proved more effective than
combined SMS treatments; as SMS treatment of
L.edodes, A.bisporus and P. sajor-caju decolorized
Chicago Sky Blue 6B in the range of 97-98% (Fig. 4),
compared with only 80-88% in combined SMS treatments.
Similar was the case with Quinaldine Red; wherein
individualized SMS supported decolorization of 86-95%,
compared with 84-86% in combined SMS treatments.
Like pH, temperature of incubation has direct role is
sustaining enzymatic activities and growth of different
microorganisms in a cultural medium, which ultimately
influences dye decolorization process. In several
studies14,16, specific temperature has been indicated for
decolorization of specific dye through a specific
microorganism. In these studies, optimum decolorization
of Reactive Red has been reported through Citrobacter
sp. CK3 at 32oC, while that of Acid red 151 through
Aspergillus niger SA1 at mesophilic temperature
(25-45 oC) and Remazol Brilliant Blue R through
Streptomyces psammoticus at 32oC. However, in none
of the cases, where mushroom species had been used
as test organism, temperature optima studies have been
conducted. In present study, variation in temperature
optima for decolorization of different dyes through SMS
of three mushrooms is mainly because of variation in
microbial population harbored by these SMS7. Excepting
few cases, decolorization was higher at 25 and 35oC,
because of 25oC as optimum temperature for vegetative
mycelial growth of three mushroom species and 25-35oC
as mesophilic temperature range suitable for dye
decolorization through mesophilic microorganisms like
1074
J SCI IND RES VOL 68 DECEMBER 2009
A. niger14, which also dominates microbial population
thrive in mushroom SMS6. In few cases, decolorization
was at par at 15, 25 and 35oC and it is attributed to 15oC
as optimum temperature for fruiting of A. bisporus and
L.edodes 20.
Present study highlighted role of three very crucial
cultural factors in decolorization of four structurally
different dyes through spent substrates of three different
mushrooms. Proper disposal of spent mushroom substrate
has always remained a challenging task for mushroom
growers’ as till now mushrooms are considered as prime
product of mushroom cultivation and left over residual
substrate which contains a variety of nutrients and
microbes is discarded. Mushroom species are grown on
different substrates (composted wheat straw + poultry
manure based substrate for A. bisporus, pasteurized
wheat straw/paddy straw substrate for P. sajor-caju
and pasteurized saw dust for L. edodes20). Although,
mushrooms have pH (7-8) and temperature optima (1528oC) for vegetative growth and fruiting, but their spent
substrates also harbor other microorganisms of mesophilic
nature, which have significant ligninolytic enzymes
activity6,18 and contribute towards dyes decolourization7.
3
Conclusions
Spent substrate from A. bisporus and L. edodes
mushroom has highest dye decolorization capacity studied
at different pH and temperature conditions of medium.
Requirement of pH of medium varied between 4-7 and
7-10 in case of SMS of different mushrooms, but in all
cases, quite high decolorization has been shown at pH
7.0, which is also ideal for substrates used in cultivation
of different mushrooms. With some exceptions, 25o and
35oC have shown quite high decolorization of different
dyes through SMS of different mushrooms; hence pH
7.0 of medium and 25oC as incubation temperature are
recommended for decolorization of different dyes through
SMS of A. bisporus and L. edodes.
12
Acknowledgements
Authors thank DST, Govt. of India for providing
financial assistance, and Director, Directorate of
Mushroom Research, Solan (HP) for requisite facilities
to carry out research work.
4
5
6
7
8
9
10
11
13
14
15
16
17
18
19
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