World Journal of Microbiology & Biotechnology 10, 556-559
Decoloration of azo dyes by three whiterot fungi: influence of carbon source W.L. Chao* and S.L. Lee Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high n i t r o g e n medium with glucose, could decolorize t w o azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). W h e n starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12 mM NH4CI, w h e t h e r or n o t veratric acid was present. In medium containing 1.2 mM NH4CI and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2 mM NH4CI, decoloration b y the local isolate was enhanced but not that b y the other strains.
Key words: Azo dye, carbon source, decoloration, white-rot fungi.
Azo dyes are used extensively in the textile and dyestuff industries. They have relatively complex structures and are resistant to microbial degradation. Some are carcinogenic or mutagenic (Cameron et al. I987). Phanerochaete chrysosporium, a major wood-rotting fungus, can degrade a wide range of recalcitrant xenobiotic compounds, including azo dyes (Aust 1990; Cripps et al. 1990), usually at a faster rate when an alternate carbon source, such as cellulose, is also present (Fernando et al. 1989). Extracellular peroxidases and laccase play an important role in the degradation of lignin and various recalcitrant compounds (Kirk & Farrell 1987; Valli et al. 1992) and these may be induced by including lignin-related compounds, such as veratryl alcohol, in the culture medium (Liebeskind et al. 1990; Lundell et al. 1990). The aim of the present study was to see if the &coloration of azo dyes by Phanerochaete spp. could be improved by pre-cultivation of the fungus with selected carbon sources and the lignin-related compound, veratric acid.
Materials and Methods Fungi and Growth Phanerochaete chrysosporium Burds., CCRC 36200 (ATCC 24715) and CCRC 36201 (ATCC 32629) were from the Culture Collection & Research Center, Food Industry Research & Development The authors are with the Department of Microbiology, Soochow University, Shih Lin, Taipei, Taiwan; fax: 886-02-8831193. *Corresponding author.
© 1994 Rapid Communications of Oxford Ltd 556
World ]ournal of Microbiology& Biotechnology,Vo110, 1994
Institute, Hsinchu, Taiwan. Strain $1 was isolated from rotten wood by using a selective medium (Dieterich & Lamar 1990). The P. chrysosporium strains were grown on 2% (w/v) malt extract/ agar at 30"C for 2 weeks. Spores were removed by suspending the cultures in distilled water and this suspension was passed through a glass-fibre filter to produce the inocula. Isolate $1 was grown on 2% malt extract/agar plates for 3 days and plugs (diam. 0.5 cm) taken from the edge served as inocula. Three media were used. Medium 1 contained (g/l): glucose, 10; ammonium tartrate, 0.22; KH~PO4, 0.9; K~HPO4, 0.1; MgSO4. 7H20, 0.5; CaC12.2H20, 0.05; thiamine, 0.001; supplemented with 10 ml micronutrient solution/1 (see below). Medium 2 was as medium 1 but with 1.64 g sodium acetate/l added and the pH adjusted to 5.0 with glacial acetic acid (final concentration 20 mM). Medium 3 was as medium 1 but with 3 ml dimethyl succinate/1 added. The micronutrient solution contained (g/1 distilled water): CuSO4.5H20, 0.08; H2MoO4, 0.05; MnSO4 4H20, 0.07; ZnSO4.7HzO, 4.3; and Fez(SO4)3,0.05. When different carbon substrates (i.e. glucose, cellulose and starch) were used for the decoloration of Orange G by the test fungi, medium 1 was used with the ammonium tartrate replaced with NH4C1.
Decoloration Study Test fungi were cultured in medium 1 at 28°C on a rotary shaker (200 rev/min) for 5 days. In some experiments, veratric acid was added at 1.5 mM. Dye solutions, 1 mE were added to the cultures and incubated as above. Triplicate flasks were prepared for each treatment and sampled daily. Each sample (1 ml) was mixed with 1 ml acetate buffer (20 mM, pH 5.0) and the absorbance at 522, 480 or 647 nm was measured to determine the concentration of Amaranth, Orange G and Azure B, respectively. Each experiment was repeated twice. All of the dyes used in this study were from Sigma.
Decoloration of azo dyes Table 1. Decoloratlon of Orange G (67 /lg/ml) by P. chryso. sporium pre-grown In different buffered media. Organism and growth medium*
Dye remaining in solution (~g/ml) after (days):
Table 2. Deceleration of dyes by white-rot fungi.* Organism and dye*
Dye remaining In solution (pg/ml) after (days): 0
5
7
10
25 40 65
4 35 62
P. chrysosporium CCRC 36200 PBM SAM DSM
38 56 66
5
7
45 38 34
12 13 13
P. chrysosporiumCCRC 36200 Amaranth Orange G Azure B
51 53 50
P. chrysosporiumCCRC 36201 P. chrysosporium CCRC 36201 PBM SAM DSM
67 67 30
49 64 12
8 64 3
* PBM--Phosphate-buffered medium 1; SAM--sodium acetatebuffered medium 2; DSM---dimethyl succinate-buffered medium 3. 50
A
Amaranth Orange G Azure B
50 51 51
47 50 47
35 49 39
Isolate $1 Amaranth Orange G Azure B
50 53 50
26 30 16
0 3 0
* The test organisms were pre-cultured in phosphate-buffered broth with glucose.
4o r
E
used in subsequent experiments. Amaranth, Orange G and Azure B were decolorized to different extents by the test fungi (Table 2). Isolate $1 caused the most rapid decoloration of all three dyes.
3O
2O v
E "o o
E
10
Influence of NH4C1 Concentration, Carbohydrates and Veratric Acid in the Pre-cultivation
O
e40 I
C
O
O~
4
5
6
® c-
30
O
2O
10
0
I
2
3
7
Time (days) Figure 1. Influence of carbon source and inducer in the precultivation medium on the decoloration of Orange G by P. chrysosporium CCRC 36200. The carbon source was (A) glucose or (B) starch. @--1.2 mM NH4CI with veratric acid; ©--1.2 mM NH,CI without veratric acid; A - - 1 2 ma NH,CI with veratric acid; A - - 1 2 mM NH4CI without veratric acid. The vertical bar gives the standard deviation from the mean. (Cellulose was not used as carbon source by this fungus.)
Results
When glucose was used as carbon source in the pre-cultivation medium containing 1.2 mM NH4CI, P. Chrysosporium did not decolorize any Orange G unless veratric acid was added and even then there was little decoloration (Figures 1A and 2A). Addition of veratric acid caused a much larger increase in the rate of decoloration by isolate $1 (Figure 3A). When pre-cultured in medium with starch as carbon source and 1.2 mM NH4C1, there was much faster decoloration of Orange G by P. chrysosporium CCRC 36200 and isolate $1 with veratric acid than without it (Figures 1B and 3B). There was no such improvement with P. chrysosporium CCRC 36201 (Figure 2B). With medium containing 12 mM NH4CI, whether or not veratric acid was added and whether glucose or starch was used as carbon source, all three fungi rapidly decolorized Orange G, P. chrysosporium CCRC 36200 being the fastest when glucose was the carbon source. With cellulose as the carbon source, both P. chrysosporium strains always failed to decolorize Orange G. Isolate $1 gave the fastest and the slowest decolorations in media containing veratric acid and 1.2 and 12 mM NH4C1, respectively (Figure 3C).
Decoloration of Orange G was highest when P. chrysosporium spp. were pre-cultured in the phosphate-buffered
Discussion
medium I (Table 1) and this medium was consequently
The decoloration of the dyes used here is likely to be by
World Journal of Microbiology & Biofechnology, Vol 10, 1994
5~ 7
W.L. Chao and 5.L. Lee 5 0 ~
50
A
.
A 40
30
2O
10
'10 O
I
I
I
I
I
I
E
B
e.
0
4oi
E 30
"o
0
E
20
20
c10
10
0 0
i
i
i
i
i
i
i
1
2
3
4
5
6
7
O')
0 Time
O
8
40
(days)
Figure 2. Influence of carbon source and inducer in the precultivation medium on the decoloration of Orange G by P. chrysosporiumCCRC 36201, The carbon source was (A) glucose or (B) starch. See legend to Figure 1 for key to symbols.
3O
2O
10
the production of various extracellular peroxidases (e.g. Lignin peroxidase and Mn-dependent peroxidase) or laccase, as established by Michel et al. (1991). Besides being affected by the type of inducer used, the production of these peroxidases is also known to depend on the growth media (Dass & Reedy 1990) and fungal strains used (Boyle et al. I992). These may very well be the reasons why different decoloration rates were observed in the present study after the test fungi were cultured in different buffered media with and without veratric acid. Cripps et al. (1990) showed that the decoloration of dyes was enhanced when P. chrososporium was pre-cultured in N-limited (1.2 mM ammonium tartrate) medium. In the present study, however, higher decoloration rates were sometimes detected when the fungi were pre-grown in a high-N medium whereas slower or no decoloration was observed when they were pre-cultured in a low-N medium. Degradation of xenobiotic compounds by some white-rot fimgi is known to occur under non-ligninolytic conditions (Dhawale et al. 1992). Although under N-sufficient conditions, P. chrysosporium did not produce lignin peroxidases and Mn-dependent peroxidases, Perle & Gold (1991) reported that laccase expression in Dichomitus squalens was activated by growth under high-N (12 mM ammonium tartrate) conditions. A specific inducer, such as veratyl alcohol, can enhance the decoloration or lignin degradation activities of white-rot fi,mgi (Faison e~ al. I986; Paszczynski & Crawford 1991). Similar results were observed when
558
World Journalof Microbiology & Biotechnology,VoI 10, 1994
0 1
2
3
Time
4
5
6
7
(days)
Figure 3. influence of carbon source and inducer in the pre-cultivation medium on the decoloration of Orange G by isolate $1. The carbon source was (A) glucose, (B) starch or (C) cellulose. See legend to Figure 1 for key to symbols.
veratric acid was used in the present study. The only exception was when isolate $1 was grown under high-N conditions with cellulose as a carbon source: here decoloration was inhibited by the presence of veratric acid. Femando et al. (1989) reported that significantly more 1,1,1-trichloro2,2-bis-(p-chlorophenyl) ethane (DDT) was mineralized by P. chrysosporium in the presence of cellulose than in the presence of glucose. The present results indicate that a similar stimulating effect of cellulose on the decoloration of Orange G is strain-dependent. It is clear that, by carefully selecting the fungal isolate and cultural conditions used, higher rates of degradation of azo dyes can be obtained.
References Aust, S.D. 1990 Degradation of environmental pollutants by Phanerochaete chrysosporium. Microbial Ecology 20, 197-209. Boyle, C.D., Kropp, B.R. & Reid, I.D. 1992 Solubilization and mineralization of lignin by white rot fungi. Applied and Environmental Microbiology 58, 3217-3224.
Decoloration of azo dyes Cameron, T.P., Hughes, T.J., Kirby, P.E., Fung, V.A. & Dunkel, V.C. 1987 Mutagenic activity of 27 dyes and related chemicals in the SalmoneIla/microsome and mouse lymphoma TK +~assays. Mutation Research 189, 223-261. Cripps, C., Bumpus, J.A. & Aust, S.D. 1990 Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Applied and Environmental Microbiology 56, 1114-1118. Dass, S.B. & Reedy, C.A. 1990 Characterization of extracellular peroxidases produced by acetate-buffered cultures of the [ignindegrading basidiomycetes Phanerochaete chrysosporium. FEMS Microbiology Letters 69, 221-224. Dhawale, S.W., Dhawale, S.S. & Dean-Ross, D. 1992 Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions. Applied and Environmental Microbiology 58, 3000-3006. Dieterich, D.M. & Lamar, R.T. 1990 Selective medium for isolation Phanerochaete chrysosporium from soil. Applied and Environmental Microbiology 56, 3088-3092. Faison, B.D., Kirk, T.K. & Farrell, R.L. 1986. Role of veratryl alcohol in regulating ligninase activity in Phanerochaetechrysosporium. Applied and Environmental Microbiology 52, 251-254. Fernando, T., AusL S.D. & Bumpus, J.A. 1989 Effects of culture parameters on DDT [1,1,1-trichloro-2,2-bis(4-chloro-phenyl)ethane] biodegradation by Phanerochaete chrysosporium. Chemosphere 19, 1387-1398. Kirk, T.K. & Farrell, R.L. 1987 Enzymatic "combustion": the microbial degradation of lignin. Annual Review of Microbiology 41, 465-505.
Liebeskind, M., Hocker, H., Wandrey, C. & Jager, A.G. 1990 Strategies for improved [ignin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer. FEMS Microbiology Letters 71, 325-330. Lundell, T., Leonowicz, A., Rogalski, J. & Hatakka, A. 1990 Formation and action of lignin-modifying enzymes in cultures of Phlebia radiata supplemented with veratric acid. Applied and Environmental Microbiology 56, 2623-2629. Michel Jr, F.C., Dass, S.B., Grulke, E.A. & Reddy, C.A. 1991 Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolourization of kraft bleach plant effluent. Applied and Environmental Microbiology 57, 23682375. Paszczynski, A. & Crawford, R.L. 1991 Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol. Biochemical and Biophysical Research Communications 178, 1056-1063. Perie, F.H. & Gold, M.H. 1991 Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens. Applied and Environment Microbiology 5 7, 2240-2245. Valli, K., Brock, B.J., Joshi, D. & Gold, M.H. 1992 Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrososporium. Applied and Environmental Microbiology 58, 221228.
(Received in revised form 4 May 1994; accepted 8 May 1994)
World Journal of Microbiology F~Biolechnology, Vol I0. 1994
559
Decoloration of azo dyes by three whiterot fungi: influence of carbon source W.L. Chao* and S.L. Lee Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high n i t r o g e n medium with glucose, could decolorize t w o azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). W h e n starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12 mM NH4CI, w h e t h e r or n o t veratric acid was present. In medium containing 1.2 mM NH4CI and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2 mM NH4CI, decoloration b y the local isolate was enhanced but not that b y the other strains.
Key words: Azo dye, carbon source, decoloration, white-rot fungi.
Azo dyes are used extensively in the textile and dyestuff industries. They have relatively complex structures and are resistant to microbial degradation. Some are carcinogenic or mutagenic (Cameron et al. I987). Phanerochaete chrysosporium, a major wood-rotting fungus, can degrade a wide range of recalcitrant xenobiotic compounds, including azo dyes (Aust 1990; Cripps et al. 1990), usually at a faster rate when an alternate carbon source, such as cellulose, is also present (Fernando et al. 1989). Extracellular peroxidases and laccase play an important role in the degradation of lignin and various recalcitrant compounds (Kirk & Farrell 1987; Valli et al. 1992) and these may be induced by including lignin-related compounds, such as veratryl alcohol, in the culture medium (Liebeskind et al. 1990; Lundell et al. 1990). The aim of the present study was to see if the &coloration of azo dyes by Phanerochaete spp. could be improved by pre-cultivation of the fungus with selected carbon sources and the lignin-related compound, veratric acid.
Materials and Methods Fungi and Growth Phanerochaete chrysosporium Burds., CCRC 36200 (ATCC 24715) and CCRC 36201 (ATCC 32629) were from the Culture Collection & Research Center, Food Industry Research & Development The authors are with the Department of Microbiology, Soochow University, Shih Lin, Taipei, Taiwan; fax: 886-02-8831193. *Corresponding author.
© 1994 Rapid Communications of Oxford Ltd 556
World ]ournal of Microbiology& Biotechnology,Vo110, 1994
Institute, Hsinchu, Taiwan. Strain $1 was isolated from rotten wood by using a selective medium (Dieterich & Lamar 1990). The P. chrysosporium strains were grown on 2% (w/v) malt extract/ agar at 30"C for 2 weeks. Spores were removed by suspending the cultures in distilled water and this suspension was passed through a glass-fibre filter to produce the inocula. Isolate $1 was grown on 2% malt extract/agar plates for 3 days and plugs (diam. 0.5 cm) taken from the edge served as inocula. Three media were used. Medium 1 contained (g/l): glucose, 10; ammonium tartrate, 0.22; KH~PO4, 0.9; K~HPO4, 0.1; MgSO4. 7H20, 0.5; CaC12.2H20, 0.05; thiamine, 0.001; supplemented with 10 ml micronutrient solution/1 (see below). Medium 2 was as medium 1 but with 1.64 g sodium acetate/l added and the pH adjusted to 5.0 with glacial acetic acid (final concentration 20 mM). Medium 3 was as medium 1 but with 3 ml dimethyl succinate/1 added. The micronutrient solution contained (g/1 distilled water): CuSO4.5H20, 0.08; H2MoO4, 0.05; MnSO4 4H20, 0.07; ZnSO4.7HzO, 4.3; and Fez(SO4)3,0.05. When different carbon substrates (i.e. glucose, cellulose and starch) were used for the decoloration of Orange G by the test fungi, medium 1 was used with the ammonium tartrate replaced with NH4C1.
Decoloration Study Test fungi were cultured in medium 1 at 28°C on a rotary shaker (200 rev/min) for 5 days. In some experiments, veratric acid was added at 1.5 mM. Dye solutions, 1 mE were added to the cultures and incubated as above. Triplicate flasks were prepared for each treatment and sampled daily. Each sample (1 ml) was mixed with 1 ml acetate buffer (20 mM, pH 5.0) and the absorbance at 522, 480 or 647 nm was measured to determine the concentration of Amaranth, Orange G and Azure B, respectively. Each experiment was repeated twice. All of the dyes used in this study were from Sigma.
Decoloration of azo dyes Table 1. Decoloratlon of Orange G (67 /lg/ml) by P. chryso. sporium pre-grown In different buffered media. Organism and growth medium*
Dye remaining in solution (~g/ml) after (days):
Table 2. Deceleration of dyes by white-rot fungi.* Organism and dye*
Dye remaining In solution (pg/ml) after (days): 0
5
7
10
25 40 65
4 35 62
P. chrysosporium CCRC 36200 PBM SAM DSM
38 56 66
5
7
45 38 34
12 13 13
P. chrysosporiumCCRC 36200 Amaranth Orange G Azure B
51 53 50
P. chrysosporiumCCRC 36201 P. chrysosporium CCRC 36201 PBM SAM DSM
67 67 30
49 64 12
8 64 3
* PBM--Phosphate-buffered medium 1; SAM--sodium acetatebuffered medium 2; DSM---dimethyl succinate-buffered medium 3. 50
A
Amaranth Orange G Azure B
50 51 51
47 50 47
35 49 39
Isolate $1 Amaranth Orange G Azure B
50 53 50
26 30 16
0 3 0
* The test organisms were pre-cultured in phosphate-buffered broth with glucose.
4o r
E
used in subsequent experiments. Amaranth, Orange G and Azure B were decolorized to different extents by the test fungi (Table 2). Isolate $1 caused the most rapid decoloration of all three dyes.
3O
2O v
E "o o
E
10
Influence of NH4C1 Concentration, Carbohydrates and Veratric Acid in the Pre-cultivation
O
e40 I
C
O
O~
4
5
6
® c-
30
O
2O
10
0
I
2
3
7
Time (days) Figure 1. Influence of carbon source and inducer in the precultivation medium on the decoloration of Orange G by P. chrysosporium CCRC 36200. The carbon source was (A) glucose or (B) starch. @--1.2 mM NH4CI with veratric acid; ©--1.2 mM NH,CI without veratric acid; A - - 1 2 ma NH,CI with veratric acid; A - - 1 2 mM NH4CI without veratric acid. The vertical bar gives the standard deviation from the mean. (Cellulose was not used as carbon source by this fungus.)
Results
When glucose was used as carbon source in the pre-cultivation medium containing 1.2 mM NH4CI, P. Chrysosporium did not decolorize any Orange G unless veratric acid was added and even then there was little decoloration (Figures 1A and 2A). Addition of veratric acid caused a much larger increase in the rate of decoloration by isolate $1 (Figure 3A). When pre-cultured in medium with starch as carbon source and 1.2 mM NH4C1, there was much faster decoloration of Orange G by P. chrysosporium CCRC 36200 and isolate $1 with veratric acid than without it (Figures 1B and 3B). There was no such improvement with P. chrysosporium CCRC 36201 (Figure 2B). With medium containing 12 mM NH4CI, whether or not veratric acid was added and whether glucose or starch was used as carbon source, all three fungi rapidly decolorized Orange G, P. chrysosporium CCRC 36200 being the fastest when glucose was the carbon source. With cellulose as the carbon source, both P. chrysosporium strains always failed to decolorize Orange G. Isolate $1 gave the fastest and the slowest decolorations in media containing veratric acid and 1.2 and 12 mM NH4C1, respectively (Figure 3C).
Decoloration of Orange G was highest when P. chrysosporium spp. were pre-cultured in the phosphate-buffered
Discussion
medium I (Table 1) and this medium was consequently
The decoloration of the dyes used here is likely to be by
World Journal of Microbiology & Biofechnology, Vol 10, 1994
5~ 7
W.L. Chao and 5.L. Lee 5 0 ~
50
A
.
A 40
30
2O
10
'10 O
I
I
I
I
I
I
E
B
e.
0
4oi
E 30
"o
0
E
20
20
c10
10
0 0
i
i
i
i
i
i
i
1
2
3
4
5
6
7
O')
0 Time
O
8
40
(days)
Figure 2. Influence of carbon source and inducer in the precultivation medium on the decoloration of Orange G by P. chrysosporiumCCRC 36201, The carbon source was (A) glucose or (B) starch. See legend to Figure 1 for key to symbols.
3O
2O
10
the production of various extracellular peroxidases (e.g. Lignin peroxidase and Mn-dependent peroxidase) or laccase, as established by Michel et al. (1991). Besides being affected by the type of inducer used, the production of these peroxidases is also known to depend on the growth media (Dass & Reedy 1990) and fungal strains used (Boyle et al. I992). These may very well be the reasons why different decoloration rates were observed in the present study after the test fungi were cultured in different buffered media with and without veratric acid. Cripps et al. (1990) showed that the decoloration of dyes was enhanced when P. chrososporium was pre-cultured in N-limited (1.2 mM ammonium tartrate) medium. In the present study, however, higher decoloration rates were sometimes detected when the fungi were pre-grown in a high-N medium whereas slower or no decoloration was observed when they were pre-cultured in a low-N medium. Degradation of xenobiotic compounds by some white-rot fimgi is known to occur under non-ligninolytic conditions (Dhawale et al. 1992). Although under N-sufficient conditions, P. chrysosporium did not produce lignin peroxidases and Mn-dependent peroxidases, Perle & Gold (1991) reported that laccase expression in Dichomitus squalens was activated by growth under high-N (12 mM ammonium tartrate) conditions. A specific inducer, such as veratyl alcohol, can enhance the decoloration or lignin degradation activities of white-rot fi,mgi (Faison e~ al. I986; Paszczynski & Crawford 1991). Similar results were observed when
558
World Journalof Microbiology & Biotechnology,VoI 10, 1994
0 1
2
3
Time
4
5
6
7
(days)
Figure 3. influence of carbon source and inducer in the pre-cultivation medium on the decoloration of Orange G by isolate $1. The carbon source was (A) glucose, (B) starch or (C) cellulose. See legend to Figure 1 for key to symbols.
veratric acid was used in the present study. The only exception was when isolate $1 was grown under high-N conditions with cellulose as a carbon source: here decoloration was inhibited by the presence of veratric acid. Femando et al. (1989) reported that significantly more 1,1,1-trichloro2,2-bis-(p-chlorophenyl) ethane (DDT) was mineralized by P. chrysosporium in the presence of cellulose than in the presence of glucose. The present results indicate that a similar stimulating effect of cellulose on the decoloration of Orange G is strain-dependent. It is clear that, by carefully selecting the fungal isolate and cultural conditions used, higher rates of degradation of azo dyes can be obtained.
References Aust, S.D. 1990 Degradation of environmental pollutants by Phanerochaete chrysosporium. Microbial Ecology 20, 197-209. Boyle, C.D., Kropp, B.R. & Reid, I.D. 1992 Solubilization and mineralization of lignin by white rot fungi. Applied and Environmental Microbiology 58, 3217-3224.
Decoloration of azo dyes Cameron, T.P., Hughes, T.J., Kirby, P.E., Fung, V.A. & Dunkel, V.C. 1987 Mutagenic activity of 27 dyes and related chemicals in the SalmoneIla/microsome and mouse lymphoma TK +~assays. Mutation Research 189, 223-261. Cripps, C., Bumpus, J.A. & Aust, S.D. 1990 Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Applied and Environmental Microbiology 56, 1114-1118. Dass, S.B. & Reedy, C.A. 1990 Characterization of extracellular peroxidases produced by acetate-buffered cultures of the [ignindegrading basidiomycetes Phanerochaete chrysosporium. FEMS Microbiology Letters 69, 221-224. Dhawale, S.W., Dhawale, S.S. & Dean-Ross, D. 1992 Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions. Applied and Environmental Microbiology 58, 3000-3006. Dieterich, D.M. & Lamar, R.T. 1990 Selective medium for isolation Phanerochaete chrysosporium from soil. Applied and Environmental Microbiology 56, 3088-3092. Faison, B.D., Kirk, T.K. & Farrell, R.L. 1986. Role of veratryl alcohol in regulating ligninase activity in Phanerochaetechrysosporium. Applied and Environmental Microbiology 52, 251-254. Fernando, T., AusL S.D. & Bumpus, J.A. 1989 Effects of culture parameters on DDT [1,1,1-trichloro-2,2-bis(4-chloro-phenyl)ethane] biodegradation by Phanerochaete chrysosporium. Chemosphere 19, 1387-1398. Kirk, T.K. & Farrell, R.L. 1987 Enzymatic "combustion": the microbial degradation of lignin. Annual Review of Microbiology 41, 465-505.
Liebeskind, M., Hocker, H., Wandrey, C. & Jager, A.G. 1990 Strategies for improved [ignin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer. FEMS Microbiology Letters 71, 325-330. Lundell, T., Leonowicz, A., Rogalski, J. & Hatakka, A. 1990 Formation and action of lignin-modifying enzymes in cultures of Phlebia radiata supplemented with veratric acid. Applied and Environmental Microbiology 56, 2623-2629. Michel Jr, F.C., Dass, S.B., Grulke, E.A. & Reddy, C.A. 1991 Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolourization of kraft bleach plant effluent. Applied and Environmental Microbiology 57, 23682375. Paszczynski, A. & Crawford, R.L. 1991 Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol. Biochemical and Biophysical Research Communications 178, 1056-1063. Perie, F.H. & Gold, M.H. 1991 Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens. Applied and Environment Microbiology 5 7, 2240-2245. Valli, K., Brock, B.J., Joshi, D. & Gold, M.H. 1992 Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrososporium. Applied and Environmental Microbiology 58, 221228.
(Received in revised form 4 May 1994; accepted 8 May 1994)
World Journal of Microbiology F~Biolechnology, Vol I0. 1994
559
