World Journal of Microbiology and Biotechnology, 8, 309-312
Biodegradation of textile azo-dyes by Phanerochaete chrysosporium N. Capalash and Prince Sharma* Of 18 commercially used textile dyes, eight were degraded by the white rot fungus, Phanerochaete chrysosporium, by 40 to 73% based on decrease of colour. Both the liguin-degrading enzyme system of P. chrysosporittm and adsorption to its cell mass were involved in the degradation of the diazo dye, Reactofix Gold Yellow. Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium; inoculation with spores resulted mainly in dye adsorption.
Key words: Biodegradation, |ignin-degrading system, Phanerochaete, textile dyes
Azo dyes constitute the largest class of dyes used commercially in the textile mills. Their mutagenic, carcinogenic and toxic potential has been extensively studied (Delclos et al. 1984; Joachim et al, 1985; Cameron et al. 1987) along with the risk of cancer associated with occupational exposure to them (Eastlander 1988, Gonazoles et al. I988; Risch et al. 1988). These dyes are released into the aquatic and terrestrial environment through the effluents emerging from textile and dye-stuff industries and are normally not removed by conventional waste-water treatment systems (Pagga & Brown 1986). The characteristic chemical structures of azo dyes, i.e. the azo linkage and aromatic sulpho group, make them recalcitrant to biological breakdown. Therefore the effluents of the textile and dyestuff industries have to be treated by physical processes which may be expensive. Attempts to isolate microorganisms which could use azo dyes as carbon and energy sources have largely been unproductive. However azo dye-degrading Pseudomonas strains have been developed using extensive chemostat cultivation (Kulla 198I). Here degradation occurred through an oxygeninsensitive azoreductase which catalysed the reductive cleavage of azo groups using NAD(P)H as an electron donor (Zimmerman et al. 1982). Various anaerobic bacteria have been reported (Meyer 1981) to degrade azo dyes by reducing the azo linkage and forming colourless and toxic N. Capalash and Prince Sharma are with the Department of Microbiology, Panjab University, Chandigarh, 160 014, India. * Corresponding author.
aromatic amines. However, under aerobic conditions, these dyes are not considered to be biodegradable. Phanerochaete chrysosporium, a ligninolytic, white-rot fungus, degrades a wide variety of structurally diverse organo-pollutants (Bumpus & Aust 1986) through its non-specific, H2Oz-dependent ' extracellular, lignin-degrading enzyme system produced during secondary metabolism in nitrogen-limited medium. This fungus has been shown to degrade azo and heterocyclic dyes (Cripps et al. 1990), Crystal Violet (Bumpus & Brock 1988) and polymeric dyes (Glenn & Gold 1983). This study reports the aerobic biodegradation of some of the commercial textile dyes by P. chrysosporium.
Materials and Methods Organism and Growth Phanerochaete chrysosporium ATCC 24725, used in this study, was grown at 25~ on 2% (w/v) malt agar and maintained at 4~ A spore inoculumwas prepared by washing the spores of the fungus from 14 day-old slants into 10 ml of sterile distilled water. The mycelial inoculum was prepared by homogenizing the 3 day-old pregrown myceliumwith sterile glass beads. It was then grown in the nitrogen-limited basal medium, prepared in 10 ram dimethyl succinate buffer,pH 4.5, as describedby Kirk et al. (1986). Liquid cultures were grown by inoculating I00/tl of spore suspension (approx 5 x 104 spores/ml) or homogenized myce]ium (1.4mg wet wt/ml) in 10 ml of basal medium contained in rubberstoppered 100-rrd Erlenmeyerflasks which were flushed with O: and incubatedat 39~ under stationary conditions.The flasks were again flushed with 02 and 0.4 mM veratryl alcohol was added on
@ 1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Biotechnology, Vol 8, I992
309
N. Capalash and Prince Sharma the third day. After 5 days, the cell-free supernatant was obtained by centrifugation of cultures at 10,000 x g for 5 rain at 4~ The culture supematant was concentrated by ultrafiltration through a membrane with a 5000 Da cut-off and used as a source of the lignin-degrading enzyme system.
N
Lignin PeroxidaseAssay The enzyme was assayed as described by Tien & Kirk (1968). One unit of enzyme activity was defined as I nmol of veratraldehyde formed per ml per rain.
Na%S
/
H
CH II CH
,..co%
,%..
Biodegradafion of Textile Dyes To 5-day-old liquid culture, stock dye solution was added in a concentration which gives an initial absorbance value of 1 at the 2m~xof the dye. Incubation was continued at 39~ samples were withdrawn at intervals, centrifuged at 10,000 x g for 5 min and the absorbance of supernatant measured. Dyes were obtained from Jagatjit Cotton Textile Mills, Punjab, India.
Effects of Extracellular Enzyme and Cell Mass Concentrations on Dye Degradation Ten ml of the dimethylsuccinate buffer (25 mM, pH 4.5), containing 0.7% (w/v) glucose and 0.5 mM dye, was supplemented with the different concentrations of extracellular lignin-degrading enzyme at a fixed concentration of cell mass or with different concentrations of cell mass at a fixed concentration of enzyme system. Incubation was continued at 39~ and the decrease in colour intensity was followed as described.
Results and Discussion Biodegradation of 18 commercial textile dyes was followed spectrophotometrically (Table 1). Eight dyes showed considerable decrease in colour by the aerobic liquid cultures of P. chrysosporium (Table 1). The absorbance decreased for 72 h and no signifcant change was observed thereafl:er. The
Figure 1. Reactofix Golden Yellow dye: 4,4'-bis[3-chloro-5-[3ureido - 4- [1,3,6- trisulphonaphthyl - 7 - azo] - phenylamino]- S- triazinylamino]-stilbene-2,2'-disulphonic acid, octa sodium salt.
results showed that these dyes, belonging to different groups, were not uniformly susceptible to microbial degradation. While the members of reactive, disperse, sulphur and vat groups were degradable those belonging to the naphthol and acid groups were not. Maximum degradation was observed in the case of Reactofix Gold Yellow dye (Figure 1) which was selected for further study. To study the role of the lignin-degrading enzyme system and cell mass of P. chrysosporium in the degradation of the Reactofix Gold Yellow dye, we observed their effect at different concentrations, singly or in concert, according to Lin et al. (1989). Glucose was added into the test system as a co-substrate to support degradation by allowing the production of H20 2 by intracellular glucose oxidase and extracellular oxidases of the cell mass. H20 2 is required for the activity of enzymes added into the system. The dye colour decreased when either the enzyme or cell mass concentrations were increased (Figures 2 and 3) indicating I 00~
Table 1. Degradation of some textile dyes added to the 5-day-old liquid cultures of Phanerochaete chrysosporlum for 72 h. Group Reactive
Disperse Sulphur Vat
Dyes* Reactofix Orange Reactofix Golden Yellow Reactofix Blue HE2R Navilene Black Sulphur Green Sulphur Red Navinon Blue Vat brown
;L,,=, (rim)
Decolorization
500 370 540 600 550 550 340 520
60 73 49 64 67 62 71 40
(%)
* Apart from Navilene Black, which is used for dying polyester, all dyes are used with cotton. The following dyes were also tested but were not degradable: Reactofix Red (reactive); Navilene Golden Yellow (disperse); Sulphur Coffee Brown (sulphur); Vat Green and Vat Orange (vat); Naphthol Base Scarlet, Naphthol ASTR, Naphthol ASBS and Naphthol Base Bordears (naphthol); Lissamyne Fast Yellow (acid).
3 10
80
World ]ournal of Microbiology and Biotechnology, Vol 8, 1992
60
=
40
O
U3
2O I
0 0
1
I
2
I
I
3 4 TIME (DAYS)
I
I
I
5
6
7
Figure 2. Effect of concentration of the lignin-degrading enzyme system on degradation of Reactofix Golden Yellow dye added into 25 mM dimethylsuccinate buffer, pH 4.5, containing 5g glucose/I, keeping cell mass concentration fixed (l.4mg/ml). Enzyme concentrations used were 0 ( I ) , 2 (O), 4 (O), 8 (&), 16 (A) and 20 (17) units. 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
Biodegradation of textile dyes that dye degradation occurred both by enzymatic action as well as adsorption to the cell mass. The concerted action of both led to greater degradation than when they occurred singly. Increasing the cell mass concentration from 0.2 to 2 mg/ml had a more pronounced effect on degradation (approx 42% increase) than had increasing the enzyme concentration from 2 to 20 U (approx 10% increase). This shows that the enzymes are probably responsible for the initial steps in dye degradation and increasing their concentration above a certain value is of little significance. This may be similar to the degradation of lignin, which is largely prevented from entering cells because of its large size. The first step of lignin degradation must be extracellular and is carried out by enzymes like lignin peroxidase, Mn-peroxidase and laccase and the degraded products then bind irreversibly to cell mass or enter the cell and become accessible to cell-bound or intracellular degradative enzymes. The rate constant of adsorption is much higher than that of degradation of mycelium-bound toxic compounds (Lin et al. 1989). To increase the degradation of dye, the medium containing the dye was inoculated with spores and decrease in colour intensity was followed. Initial colour decrease was rapid for the first 4 days and then slowed (Figure 4). The fungal mycelium did not form a proper mat; only a broken mass could be seen, which may have been responsible for more adsorption of dye because of increased surface area. Since the dye inhibited fungal growth using a spore inoculum, the medium containing the dye was inoculated with pre-grown vegetative cells. A control set of flasks was inoculated with the same but autoclaved mycelium. The initial fall in absorbance was considerable and was similar
~
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80
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< 0
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4O 2o 0
0
i
I
I
I
,
I
i
1
2
3
4
5
6
7
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(DAYS)
Figure 4. Degradation of Reactofix Golden Yellow dye added into the medium at the time of inoculation with approx 5 x 106 spores/ml ([~) or live (O) or dead (Q) mycelium (1.4 mg/ml). The control had no inoculation (A). 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
for 3 days with both the live and dead mycelia. Although no further decrease in colour was observed after day 3 with the killed mycelium, there was a substantial decrease (approx 90%) in colour up to day 5 with the live mycelium, indicating the degradation by the lignin-degrading enzyme system, produced during secondary metabolism. Biodegradation of dyes can, therefore, be increased by further improving the initial cultural conditions of the mould. Paszczynski et aL (1991) improved the degradation of azo dyes by P, chrysosporium by introducing a metabolizable guaiacol substituent into the chemical structure of the dye, thus making it more susceptible to degradation by lignin-degrading enzymes. The non-specific, lignin-degrading system of P. chrysosporium can be significantly utilized for the aerobic degradation of heterogeneous mixtures of recalcitrant dyes in waste waters. Compared to this, bacterial degradation suffers the limitations of specificity, anaerobic conditions and very slow degradation rates, which render any bacterial strain to be of no practical value for degrading the mixture of dyes in textile mill effluent.
60 References
40 9 20 ,< 0
0
,
,
,
i
I
i
,
1
2
3
4
5
6
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TIME (DAYS) Figure 3. Effect of cell mass concentration on degradation of Reactofix Golden Yellow dye added into 25mM dimethylsuccinate buffer, pH 4.5, containing 7 g glucose/I, keeping lignin-degrading enzyme concentration fixed at 5units. Cell mass concentrations used were 0 ( x ), 0.2 ( 9 0.4 (O), 0.8 (A), 1.2 (A), 1.6 ([]) and 2.0 ( I ) mg/ml. 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
Bumpus, J.A. & Aust, S.D. 1986 Biodegradation of environmental pollutants by the white-rot fungus Phanerochaetechrysosporium: involvement of the lignin degrading system. Bio-Essays 6, 166--170. Bumpus, J.A. & Brock, B.J. 1988 Biodegradation of crystal violet by the white rot fungus Phanerochaetechrysosporium. Applied and Environmental Microbiology 54, 1143-1150. 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 Salmonella/microsome and mouse lymphoma TK+Lassays. 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 EnvironmentalMicrobiology 56, 1114-1118.
World Journal of Microbiology and Biotechnology, Vol 8, 1992
311
N. Capalash and Prince Sharma Delclos, K.B., Tarpley, W.G., Miller, E.C. & Miller, J.A. 1984 4-Aminoazobenzene and N,N-dimethyl-4-aminoazobenzene as equipotent hepatic carcinogens in male C57BL/6XC3H/He Ft mice and characterization of N-(deoxyguanosine-8-yl)-4aminoazobenzene as the major persistent hepatic DNA-bound dye in these mice. Cancer Research 44, 2540--2550. Eastlander, T. 1988 Allergic dermatoses and respiratory diseases from reactive dyes. Contact Dermatitis 18, 290--297. Glenn, J.K. & Gold, M.H. 1983 Decolourization of several po[ymeric dyes by the lignin degrading basidiomycete Phanerochaetechrysosporium. Applied and Environmental Microbiology 45, 1741-1747. Gonazoles, C.A., Elio, R. & Gonzalo, L.B. 1988 Bladder cancer among workers in the textile industry: Results of a Spanish case control study. American Journal of Industrial Medicine 14, 673-680. Joachim, F., BurrelL A. & Anderson, J. 1985 Mutagenicity of azo dyes in the SalraoneIla/microsome assay using in vitro and in vivo activation. Mutation Research 156, 131-138. Kirk, T.K., Croan, S., Tien, M., Murtagah, K.E, & Farrell, R.L. 1986 Production of multiple ligninases by Phanerochaetechrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme and Microbial Technology 8, 27-32. Kul[a, H.G. 1981 Aerobic bacterial degradation of azo dyes. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds, eds Leisinger, T., Cook, A.M., Nuesch, J. & Hutter, R. pp 387-399. London: Academic Press.
~1 ~-
World]ourr~lof Microbiologyand Biotechnology,Vol 8, 1992
Lin, J., Wang, H.Y. & Hickey, R.F. 1990 Degradation kinetics of pentachlorophenol by Phanerochaetechrysosporium. Biotechnology and Bioengineering 35, 1125-1134. Meyer, U. 198I Biodegradation of synthetic organic colourants. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds, eds Leisinger, T., Cook, A.M., Nuesch, J. & Hurter, R. pp 371-385. London: Academic Press. Pagga, U. & Brown, D. 1986 The degradation of dyestuffs. II. Behaviour of dyestuffs in aerobic biodegradation tests. Chemosphere 15, 479-491. Paszczynski, A., Pasti, M,B., Goszczynski, S., Crawford, D.L. & Crawford, R.L. 1991 New approach to improve degradation of recalcitrant azo dyes by Streptomyces slop. and Phanerochaete chrysosporium. Enzyme and Microbial Technolog!/ 13, 378--384. Risch, H.A., Burch, J.D., Miller, A.B., Hill, G.B., Steele, R. & Howe, G.R. 1988 Occupational factors and the incidence of cancer of the bladder in Canada. British Journal of Industrial Medicine 46, 361-307. Tien, M. & Kirk, T.K. 1988 Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymology 161, 238--249. Zimmerman, T., Kulla, H.G. & Leisinger, T. 1982 Properties of purified Orange II azoreductase; the enzyme initiating azo dye degradation by Pseudomonas KF 46. European Journal of Biochemishay 129, 197-203.
{Received 9 January I992; accepted I9 January 1992)
Biodegradation of textile azo-dyes by Phanerochaete chrysosporium N. Capalash and Prince Sharma* Of 18 commercially used textile dyes, eight were degraded by the white rot fungus, Phanerochaete chrysosporium, by 40 to 73% based on decrease of colour. Both the liguin-degrading enzyme system of P. chrysosporittm and adsorption to its cell mass were involved in the degradation of the diazo dye, Reactofix Gold Yellow. Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium; inoculation with spores resulted mainly in dye adsorption.
Key words: Biodegradation, |ignin-degrading system, Phanerochaete, textile dyes
Azo dyes constitute the largest class of dyes used commercially in the textile mills. Their mutagenic, carcinogenic and toxic potential has been extensively studied (Delclos et al. 1984; Joachim et al, 1985; Cameron et al. 1987) along with the risk of cancer associated with occupational exposure to them (Eastlander 1988, Gonazoles et al. I988; Risch et al. 1988). These dyes are released into the aquatic and terrestrial environment through the effluents emerging from textile and dye-stuff industries and are normally not removed by conventional waste-water treatment systems (Pagga & Brown 1986). The characteristic chemical structures of azo dyes, i.e. the azo linkage and aromatic sulpho group, make them recalcitrant to biological breakdown. Therefore the effluents of the textile and dyestuff industries have to be treated by physical processes which may be expensive. Attempts to isolate microorganisms which could use azo dyes as carbon and energy sources have largely been unproductive. However azo dye-degrading Pseudomonas strains have been developed using extensive chemostat cultivation (Kulla 198I). Here degradation occurred through an oxygeninsensitive azoreductase which catalysed the reductive cleavage of azo groups using NAD(P)H as an electron donor (Zimmerman et al. 1982). Various anaerobic bacteria have been reported (Meyer 1981) to degrade azo dyes by reducing the azo linkage and forming colourless and toxic N. Capalash and Prince Sharma are with the Department of Microbiology, Panjab University, Chandigarh, 160 014, India. * Corresponding author.
aromatic amines. However, under aerobic conditions, these dyes are not considered to be biodegradable. Phanerochaete chrysosporium, a ligninolytic, white-rot fungus, degrades a wide variety of structurally diverse organo-pollutants (Bumpus & Aust 1986) through its non-specific, H2Oz-dependent ' extracellular, lignin-degrading enzyme system produced during secondary metabolism in nitrogen-limited medium. This fungus has been shown to degrade azo and heterocyclic dyes (Cripps et al. 1990), Crystal Violet (Bumpus & Brock 1988) and polymeric dyes (Glenn & Gold 1983). This study reports the aerobic biodegradation of some of the commercial textile dyes by P. chrysosporium.
Materials and Methods Organism and Growth Phanerochaete chrysosporium ATCC 24725, used in this study, was grown at 25~ on 2% (w/v) malt agar and maintained at 4~ A spore inoculumwas prepared by washing the spores of the fungus from 14 day-old slants into 10 ml of sterile distilled water. The mycelial inoculum was prepared by homogenizing the 3 day-old pregrown myceliumwith sterile glass beads. It was then grown in the nitrogen-limited basal medium, prepared in 10 ram dimethyl succinate buffer,pH 4.5, as describedby Kirk et al. (1986). Liquid cultures were grown by inoculating I00/tl of spore suspension (approx 5 x 104 spores/ml) or homogenized myce]ium (1.4mg wet wt/ml) in 10 ml of basal medium contained in rubberstoppered 100-rrd Erlenmeyerflasks which were flushed with O: and incubatedat 39~ under stationary conditions.The flasks were again flushed with 02 and 0.4 mM veratryl alcohol was added on
@ 1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Biotechnology, Vol 8, I992
309
N. Capalash and Prince Sharma the third day. After 5 days, the cell-free supernatant was obtained by centrifugation of cultures at 10,000 x g for 5 rain at 4~ The culture supematant was concentrated by ultrafiltration through a membrane with a 5000 Da cut-off and used as a source of the lignin-degrading enzyme system.
N
Lignin PeroxidaseAssay The enzyme was assayed as described by Tien & Kirk (1968). One unit of enzyme activity was defined as I nmol of veratraldehyde formed per ml per rain.
Na%S
/
H
CH II CH
,..co%
,%..
Biodegradafion of Textile Dyes To 5-day-old liquid culture, stock dye solution was added in a concentration which gives an initial absorbance value of 1 at the 2m~xof the dye. Incubation was continued at 39~ samples were withdrawn at intervals, centrifuged at 10,000 x g for 5 min and the absorbance of supernatant measured. Dyes were obtained from Jagatjit Cotton Textile Mills, Punjab, India.
Effects of Extracellular Enzyme and Cell Mass Concentrations on Dye Degradation Ten ml of the dimethylsuccinate buffer (25 mM, pH 4.5), containing 0.7% (w/v) glucose and 0.5 mM dye, was supplemented with the different concentrations of extracellular lignin-degrading enzyme at a fixed concentration of cell mass or with different concentrations of cell mass at a fixed concentration of enzyme system. Incubation was continued at 39~ and the decrease in colour intensity was followed as described.
Results and Discussion Biodegradation of 18 commercial textile dyes was followed spectrophotometrically (Table 1). Eight dyes showed considerable decrease in colour by the aerobic liquid cultures of P. chrysosporium (Table 1). The absorbance decreased for 72 h and no signifcant change was observed thereafl:er. The
Figure 1. Reactofix Golden Yellow dye: 4,4'-bis[3-chloro-5-[3ureido - 4- [1,3,6- trisulphonaphthyl - 7 - azo] - phenylamino]- S- triazinylamino]-stilbene-2,2'-disulphonic acid, octa sodium salt.
results showed that these dyes, belonging to different groups, were not uniformly susceptible to microbial degradation. While the members of reactive, disperse, sulphur and vat groups were degradable those belonging to the naphthol and acid groups were not. Maximum degradation was observed in the case of Reactofix Gold Yellow dye (Figure 1) which was selected for further study. To study the role of the lignin-degrading enzyme system and cell mass of P. chrysosporium in the degradation of the Reactofix Gold Yellow dye, we observed their effect at different concentrations, singly or in concert, according to Lin et al. (1989). Glucose was added into the test system as a co-substrate to support degradation by allowing the production of H20 2 by intracellular glucose oxidase and extracellular oxidases of the cell mass. H20 2 is required for the activity of enzymes added into the system. The dye colour decreased when either the enzyme or cell mass concentrations were increased (Figures 2 and 3) indicating I 00~
Table 1. Degradation of some textile dyes added to the 5-day-old liquid cultures of Phanerochaete chrysosporlum for 72 h. Group Reactive
Disperse Sulphur Vat
Dyes* Reactofix Orange Reactofix Golden Yellow Reactofix Blue HE2R Navilene Black Sulphur Green Sulphur Red Navinon Blue Vat brown
;L,,=, (rim)
Decolorization
500 370 540 600 550 550 340 520
60 73 49 64 67 62 71 40
(%)
* Apart from Navilene Black, which is used for dying polyester, all dyes are used with cotton. The following dyes were also tested but were not degradable: Reactofix Red (reactive); Navilene Golden Yellow (disperse); Sulphur Coffee Brown (sulphur); Vat Green and Vat Orange (vat); Naphthol Base Scarlet, Naphthol ASTR, Naphthol ASBS and Naphthol Base Bordears (naphthol); Lissamyne Fast Yellow (acid).
3 10
80
World ]ournal of Microbiology and Biotechnology, Vol 8, 1992
60
=
40
O
U3
2O I
0 0
1
I
2
I
I
3 4 TIME (DAYS)
I
I
I
5
6
7
Figure 2. Effect of concentration of the lignin-degrading enzyme system on degradation of Reactofix Golden Yellow dye added into 25 mM dimethylsuccinate buffer, pH 4.5, containing 5g glucose/I, keeping cell mass concentration fixed (l.4mg/ml). Enzyme concentrations used were 0 ( I ) , 2 (O), 4 (O), 8 (&), 16 (A) and 20 (17) units. 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
Biodegradation of textile dyes that dye degradation occurred both by enzymatic action as well as adsorption to the cell mass. The concerted action of both led to greater degradation than when they occurred singly. Increasing the cell mass concentration from 0.2 to 2 mg/ml had a more pronounced effect on degradation (approx 42% increase) than had increasing the enzyme concentration from 2 to 20 U (approx 10% increase). This shows that the enzymes are probably responsible for the initial steps in dye degradation and increasing their concentration above a certain value is of little significance. This may be similar to the degradation of lignin, which is largely prevented from entering cells because of its large size. The first step of lignin degradation must be extracellular and is carried out by enzymes like lignin peroxidase, Mn-peroxidase and laccase and the degraded products then bind irreversibly to cell mass or enter the cell and become accessible to cell-bound or intracellular degradative enzymes. The rate constant of adsorption is much higher than that of degradation of mycelium-bound toxic compounds (Lin et al. 1989). To increase the degradation of dye, the medium containing the dye was inoculated with spores and decrease in colour intensity was followed. Initial colour decrease was rapid for the first 4 days and then slowed (Figure 4). The fungal mycelium did not form a proper mat; only a broken mass could be seen, which may have been responsible for more adsorption of dye because of increased surface area. Since the dye inhibited fungal growth using a spore inoculum, the medium containing the dye was inoculated with pre-grown vegetative cells. A control set of flasks was inoculated with the same but autoclaved mycelium. The initial fall in absorbance was considerable and was similar
~
t 00' 80
" " 1 O0 r--
80
'"
60
a
Z
< 0
m N
4O 2o 0
0
i
I
I
I
,
I
i
1
2
3
4
5
6
7
TIME
(DAYS)
Figure 4. Degradation of Reactofix Golden Yellow dye added into the medium at the time of inoculation with approx 5 x 106 spores/ml ([~) or live (O) or dead (Q) mycelium (1.4 mg/ml). The control had no inoculation (A). 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
for 3 days with both the live and dead mycelia. Although no further decrease in colour was observed after day 3 with the killed mycelium, there was a substantial decrease (approx 90%) in colour up to day 5 with the live mycelium, indicating the degradation by the lignin-degrading enzyme system, produced during secondary metabolism. Biodegradation of dyes can, therefore, be increased by further improving the initial cultural conditions of the mould. Paszczynski et aL (1991) improved the degradation of azo dyes by P, chrysosporium by introducing a metabolizable guaiacol substituent into the chemical structure of the dye, thus making it more susceptible to degradation by lignin-degrading enzymes. The non-specific, lignin-degrading system of P. chrysosporium can be significantly utilized for the aerobic degradation of heterogeneous mixtures of recalcitrant dyes in waste waters. Compared to this, bacterial degradation suffers the limitations of specificity, anaerobic conditions and very slow degradation rates, which render any bacterial strain to be of no practical value for degrading the mixture of dyes in textile mill effluent.
60 References
40 9 20 ,< 0
0
,
,
,
i
I
i
,
1
2
3
4
5
6
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TIME (DAYS) Figure 3. Effect of cell mass concentration on degradation of Reactofix Golden Yellow dye added into 25mM dimethylsuccinate buffer, pH 4.5, containing 7 g glucose/I, keeping lignin-degrading enzyme concentration fixed at 5units. Cell mass concentrations used were 0 ( x ), 0.2 ( 9 0.4 (O), 0.8 (A), 1.2 (A), 1.6 ([]) and 2.0 ( I ) mg/ml. 100% absorbance corresponds to an absorbance value at 370 nm of 1.0.
Bumpus, J.A. & Aust, S.D. 1986 Biodegradation of environmental pollutants by the white-rot fungus Phanerochaetechrysosporium: involvement of the lignin degrading system. Bio-Essays 6, 166--170. Bumpus, J.A. & Brock, B.J. 1988 Biodegradation of crystal violet by the white rot fungus Phanerochaetechrysosporium. Applied and Environmental Microbiology 54, 1143-1150. 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 Salmonella/microsome and mouse lymphoma TK+Lassays. 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 EnvironmentalMicrobiology 56, 1114-1118.
World Journal of Microbiology and Biotechnology, Vol 8, 1992
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N. Capalash and Prince Sharma Delclos, K.B., Tarpley, W.G., Miller, E.C. & Miller, J.A. 1984 4-Aminoazobenzene and N,N-dimethyl-4-aminoazobenzene as equipotent hepatic carcinogens in male C57BL/6XC3H/He Ft mice and characterization of N-(deoxyguanosine-8-yl)-4aminoazobenzene as the major persistent hepatic DNA-bound dye in these mice. Cancer Research 44, 2540--2550. Eastlander, T. 1988 Allergic dermatoses and respiratory diseases from reactive dyes. Contact Dermatitis 18, 290--297. Glenn, J.K. & Gold, M.H. 1983 Decolourization of several po[ymeric dyes by the lignin degrading basidiomycete Phanerochaetechrysosporium. Applied and Environmental Microbiology 45, 1741-1747. Gonazoles, C.A., Elio, R. & Gonzalo, L.B. 1988 Bladder cancer among workers in the textile industry: Results of a Spanish case control study. American Journal of Industrial Medicine 14, 673-680. Joachim, F., BurrelL A. & Anderson, J. 1985 Mutagenicity of azo dyes in the SalraoneIla/microsome assay using in vitro and in vivo activation. Mutation Research 156, 131-138. Kirk, T.K., Croan, S., Tien, M., Murtagah, K.E, & Farrell, R.L. 1986 Production of multiple ligninases by Phanerochaetechrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme and Microbial Technology 8, 27-32. Kul[a, H.G. 1981 Aerobic bacterial degradation of azo dyes. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds, eds Leisinger, T., Cook, A.M., Nuesch, J. & Hutter, R. pp 387-399. London: Academic Press.
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World]ourr~lof Microbiologyand Biotechnology,Vol 8, 1992
Lin, J., Wang, H.Y. & Hickey, R.F. 1990 Degradation kinetics of pentachlorophenol by Phanerochaetechrysosporium. Biotechnology and Bioengineering 35, 1125-1134. Meyer, U. 198I Biodegradation of synthetic organic colourants. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds, eds Leisinger, T., Cook, A.M., Nuesch, J. & Hurter, R. pp 371-385. London: Academic Press. Pagga, U. & Brown, D. 1986 The degradation of dyestuffs. II. Behaviour of dyestuffs in aerobic biodegradation tests. Chemosphere 15, 479-491. Paszczynski, A., Pasti, M,B., Goszczynski, S., Crawford, D.L. & Crawford, R.L. 1991 New approach to improve degradation of recalcitrant azo dyes by Streptomyces slop. and Phanerochaete chrysosporium. Enzyme and Microbial Technolog!/ 13, 378--384. Risch, H.A., Burch, J.D., Miller, A.B., Hill, G.B., Steele, R. & Howe, G.R. 1988 Occupational factors and the incidence of cancer of the bladder in Canada. British Journal of Industrial Medicine 46, 361-307. Tien, M. & Kirk, T.K. 1988 Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymology 161, 238--249. Zimmerman, T., Kulla, H.G. & Leisinger, T. 1982 Properties of purified Orange II azoreductase; the enzyme initiating azo dye degradation by Pseudomonas KF 46. European Journal of Biochemishay 129, 197-203.
{Received 9 January I992; accepted I9 January 1992)
