Enzyme and Microbial Technology 29 (2001) 575–579

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Studies on the production of enzymes by white-rot fungi for the
decolourisation of textile dyes
T. Robinson, B. Chandran, P. Nigam*
School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, UK
Received 24 May 2001; received in revised form 24 July 2001; accepted 31 July 2001

Abstract
Four white- rot fungi, Bjerkandera adusta, Phlebia tremellosa, Pleurotus ostreatus and Coriolus versicolor, were tested for their ability
to produce Lignin Peroxidase (LiP), Manganese Peroxidase (MnP), and Laccase in a nitrogen deficient mineral salts medium. B. adusta and
P. tremellosa were selected, due to their high peak enzyme activities, for the degradation of 5 dyes in an artificial textile-effluent.
Degradation experiments were carried out in N-rich (C:N ratio, 11.6:1) and N-limited, 116:1) conditions at a dye concentration of 100
mg/liter. B. adusta degraded 85% of the dyes in 7 days and P. tremellosa 79% in 9 days in N-rich media. 86% of the effluent was degraded
in 9 days by B. adusta and 74% by P. tremellosa in 11 days in N-limited conditions. Addition of nitrogen had no substantial effect on
percentage dye degradation by B. adusta, with a slight increase for P. tremellosa. Nitrogen supplementation did however reduce the decolourisation time. The results show the potential of using B. adusta and P. tremellosa for textile- effluent degradation, with or without nitrogen
supplementation.
Keywords: Degradation; Laccase; LiP; MnP; Textile-dye effluent; White-rot fungi

1. Introduction
Lignin Peroxiadse (LiP), Manganese Peroxidase (MnP)
and Laccase are the major enzymes associated with the
lignin- degrading capabilities of white-rot fungi [1]. These
non-specific enzymes which allow white-rot fungi to degrade lignin, also allows them to degrade a wide range of
pollutants. This has led to research regarding their use for
the degradation of recalcitrant compounds, such as, polycyclic aromatic hydrocarbons (PAH’s) and dyes [2,3].
There are more than 100,000 commercial dyes available
to textile industries who consume vast quantities of water
and chemicals during processing, ultimately producing dyecontaining effluent which consists of complex, synthetic
and often recalcitrant compounds. Dyes are designed to be
resistant to light, water and oxidising agents and are therefore difficult to degrade once released into aquatic systems
[4,5]. Conventional treatments of textile effluents are either
ineffective, costly, complicated or have sludge disposal

* Corresponding author. P. Nigam (Singh) Tel.: ⫹44-28-7032-4053;
fax: ⫹44-28-7032-4965.
E-mail address: P.Nigam@ulst.ac.uk (P. Nigam).

problems, so the potential exists for the breakdown of dyes
in effluent by extracellular enzymes produced by white-rot
fungi during fermentation.
This study assessed the enzyme activities of four whiterot fungi in a nitrogen deficient mineral salts medium. From
this two were selected for their potential to degrade 5 dyes
in an artificial textile- effluent in both N-rich and N-limited
conditions.

2. Materials and methods
2.1. Chemicals
All the chemicals used were reagent grade unless other
wise stated. Cibacron Yellow C-2R, Cibacron Red C-2G,
Cibacron Blue C-R, Remazol Black B and Remazol Red RB
were a gift from Fruit of the Loom, Buncrana, Rep. of
Ireland. The dyes were added in equal quantities (200 mg/
liter) to distilled water to produce an artificial effluent at a
concentration of 1000 mg/liter. A concentration of 100
mg/liter was used for dye decolourisation experiments.

0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 4 3 0 - 6

5 mM). with P.6:1 compared to 116:1 for N-rich media. MgSO4 䡠 7H2O (0. It had the following composition: KH2PO4 (0. From this. was also used for dye decolourisation experiments. ZnSO4 䡠 7H2O (43 mg/liter) and Fe(SO4)3 (50 mg/liter) and was filter sterilised prior to use.4 0. adusta had the highest production of LiP. Experiments were carried out in duplicate for both N-rich and N-limiting conditions. and activities reported as UL⫺1. Canada. Bjerkandera adusta. with MnP being produced in the smallest quantities. Enzyme activities generally began between days 1 and 3. Alberta. with peak activities between days 6 and 9. MnP and laccase activity. P.58 38. B.2-dimethylsuccinic acid (2. thiamine (0. [9].2% peptone added to produce a C:N ratio of 11. The trace elements solution contained: CuSO4 䡠 7H2O (80 mg/liter). Decolourisation of textile-effluent in N-rich medium B. Enzyme activity experiments 10 mm plugs were taken from stock plates and were inoculated into 50 ml of Malt Extract Broth. tremellosa was described by Reid [7]. The mineral salts media used for P.9 1.6 9 9 4 83. 2. 5 ml of the homogenised mycelia was then transferred to 45 ml of Kirks media (and P. CaCl2 (0. and the supernatant used to measure enzyme activities in a spectrophotometer.9 98. Tween 80 (0. Dye decolourisation experiments 5. 3. MnSO4 (33 mg/liter). Kirks media. Pleurotus ostreatus. ostreatus and C. LiP. versicolor Peak UL Day Peak UL Day Peak UL Day Peak UL⫺1 Day 170.22 g/l). adusta produced a decrease in the textile-dye effluent with a corresponding increase in enzyme activity over the period of fermentation. All cultures were obtained from the Canadian Culture Collection. ammonium tartrate (0. Enzyme assays All enzymes were determined spectrophotomerically.5. tremellosa grown on the specialised media and Kirks media.3. two fungi were selected for their ability to produce enzymes and decolourise an artificial textile-effluent consisting of 5 dyes.ml of homogenised mycelium from each fungus were added.59 32. Coriolous versicolor. All fungi had LiP. ostreatus C. Laccase activity was determined by the method of Szklarz et al.4 6 12 8 101. adusta ⫺1 LiP MnP Laccase P.8 6 9 8 98. trace elements (10.1 8 6 11 2.0 ml/liter). Phlebia tremellosa. Fungi were maintained on PDA plates and stored at 4°C and subcultured every month.3 5. versicolor produced 100 and 82 UL⫺1 of LiP respectively. tremellosa ⫺1 P. 3. but the lowest of the 4 regarding laccase activities. veratryl alcohol (1. White-rot fungi The following white-rot fungi were used. H2MoO4 (50 mg/liter). adusta had the highest MnP activities of the 4 white-rot fungi. / Enzyme and Microbial Technology 29 (2001) 575–579 Table 1 Peak enzyme activities on N-limiting Kirk’s medium (without artificial effluent) B.90 g/l).20 g/l).576 T. One unit of enzyme activity was defined as 1 ␮M of veratryl alcohol or quinone oxidised in 1 min under defined conditions. MnP activity was measured by the method of Paszczynski et al. Robinson et al. 2. with 5 ml of 1000 mg/liter of artificial effluent stock solution. MnP and laccase activities The media used for measurement of enzyme activity was a nitrogen deficient mineral salts Kirks medium. tremellosa media). tremellosa and B.01 g/l). as described by Kirby [6]. although activity increased during periods of dye deg- . [10]. LiP activity was performed according to the method of Tien and Kirk [8].2. Media compositions 3. 2. 1a). so Kirks was used for the decolourisation experiments. Samples were taken at regular intervals. P. This was carried out in duplicate for each fungus and incubated statically at optimal temperatures for each culture for 5 days. The fungi were again incubated statically at the various optimal temperatures. as described above.10 g/l).1. but only small amounts of MnP (Table 1). with LiP activity peaking at the time of maximal degradation (Fig.2.10% v/v). Laccase activity fluctuated.6.1 20. ⫺1 Dye decolourisation experiments were carried out in N-rich and N-limiting conditions.2 3. tremellosa having the highest laccase activity.05 g/l). with 0. Similar enzyme activities were found for P. Results 2. centrifuged. to 40 ml of Kirks media. The following were added post autoclaving: glucose (10 g/l). 2.4. After 5 days excess media was drained and the fungal mycelia macerated.

T. --Œ-.MnP. 3. with a slightly higher degree of degradation. --⫻-. with a high initial production after day 1. radation. tremellosa successfully degraded 79% of the artificial effluent after 9 days. adusta. 1. tremellosa. / Enzyme and Microbial Technology 29 (2001) 575–579 577 Fig.laccase. —■— effluent decolourisation (%). --䉬-MnP. Higher activity levels of LiP were maintained for both organisms throughout the fermentation period in Nlimited conditions. corresponding to a decline in the dye concentrations left in the effluent (Fig.MnP.3. A lag phase of degradation occurred at the beginning of the fermentation in both nitrogen levels.LiP. P. --⫻-. adusta degraded a similar amount of textile-effluent in N-rich media (Fig. tremellosa degraded 74% of the artificial-effluent in 11 days in N-limited conditions. the amount of enzyme activity under N-rich conditions was much higher. --Œ-. but a longer lag phase was recorded in the N-limiting conditions. Robinson et al. --Œ-. tremellosa. --䉬-MnP.LiP. B. There were sharp increases in LiP and MnP activities between days 7 and 9. 1b). LiP. Al- though the amount of dye degraded was similar.LiP. . --Œ-. (B) Production of lignolytic enzymes and textile-effluent decolourisation in N-rich media by P. from day 1–5 for both fungi after which most of the dyes were degraded. (A) Production of lignolytic enzymes and textile-effluent decolourisation in N-rich media by B. Decolourisation of textile-effluent in N-limited medium In N-limited conditions B. —■— effluent decolourisation (%). 2a).laccase. —■— effluent decolourisation (%).laccase.laccase. --䉬-. adusta. (A) Production o f lignolytic enzymes and textile-effluent decolourisation in N-limited media by B. --⫻--. P. (B) Production of lignolytic enzymes and textile-effluent decolourisation in N-limited media by P. adusta degraded 85% of the dyes in N-rich conditions in 9 days with a similar amount of Fig. 2. —■— effluent decolourisation (%). --⫻-. A total of 85% of the dyes in the artificial effluent were degraded and a period of 7 days was required for the achieved decolourisation through degradation of the dye compounds. --䉬-.

the higher enzyme activities seem to have had no substantial effect on degrading any of the dye effluent further. The major enzymes involved in dye degradation by B. LiP and laccase showed the biggest increase of the 3 enzymes during the degradation P. Robinson et al. Assistance in the decolourisation of the effluent may be credited to other enzymes present in the culture supernatant and mycelia bound enzymes. adusta.9 22.74 0. The main enzymes that appear to be used for dye degradation are LiP and laccase. B.6 79. Similarly to P. Laccase activity by P. This study produced a better percentage decolourisation through dye degradation in a shorter fermentation time with higher enzyme activities. which were not assayed for.9% 112. Whether or not the additional nitrogen should be added to increase degradation slightly is very much dependant on cost. Kirby [6]. There was in fact a small decline in LiP activity. adusta were grown in the presence of 100 mg/liter of the artificial effluent at both high and low N levels. tremellosa LiP MnP Laccase % decolourisation B. using the current assay.5 73. it cannot be concluded if MnP played any role in aiding dye decolourisation. 2b). tremellosa and B. tremellosa.7 UL⫺1. as they were produced in the highest quantities with and without the presence of dyes and at the two different nitrogen conditions.4. 70% degradation over a period of 14 days. with a smaller increase in N-limited conditions. with a slight fall in activity when grown in N-limited media.4 218. The amount of LiP increased in the presence of the artificial textile-effluent in both nitrogen conditions for P. 114. The LiP activity did not increase as dramatically in the presence of the dyes in B. adusta occured rapidly over a period of days in both N-rich and N-limited conditions.72 13.7% of the effluent. although the percentage of dye degradation was very similar in both nitrogen conditions. tremellosa was capable of decolourising a different artificial effluent. Although the percentage decolourisation is 5% higher in the N-rich media. Degradation by B. In N-limited conditions laccase activity was doubled. In this study degradation in both N-rich and N-limited conditions occurred gradually over a period of 9 days. Although the amounts of MnP were low. adusta are also LiP and laccase. [11].9% 132. tremellosa in the presence of dyes and at the different nitrogen conditions. adusta showed a slight decrease in LiP production when grown in dye and both nitrogen varying media. ostreatus and C. adusta LiP MnP Laccase % decolourisation N-rich fermentation (UL⫺1) N-limited fermentation (UL⫺1) 179.9 3. Although there was an increase in enzyme produc- . suggesting that the presence of dyes and the nitrogen supplement led to an increase in laccase production. adusta were selected for their high enzyme activities. with almost twice as much laccase being produced in N-rich conditions compared to that produced in N-limited conditions (Table 2). This also agrees with the enzyme activities of P.578 T. Effect of dyes and nitrogen content on enzyme activities P. higher enzyme activities were observed in N-rich media compared to N-limited media. The results concerning P. N-rich media.5 85. N-rich media had the effect of causing dye degradation to begin earlier. tremellosa at the two nitrogen conditions.9 UL⫺1 in N-rich media.1% 114. They found that higher ligninolytic activities were recorded in an enriched nitrogen medium.. tremellosa. tremellosa and B.93 390. An increase in laccase activity was noted in N-rich conditions. adusta and P. MnP activity was reduced by a quarter in both nitrogen conditions.6 84.9 0. with most dye breakdown occurring between days 3–7 in N-rich media and between days 5–9 in N-limited media. tremellosa increased dramatically (44%) in N-rich media. P. Table 2 Effect of nitrogen conditions on enzyme synthesis and textile-effluent decolourisation Fungi and enzymes 3. seem to indicate that laccase is one of the major ligninolytic enzymes involved in the breakdown of the dyes in the artificial effluent. tremellosa. versicolor did not produce the same high enyme activities for LiP and laccase and so were not used for the textile-effluent degradation experiments. showed that P. after day 3. although laccase levels were increased 4 fold in the presence of the artificial effluent in N-rich media. Discussion P. The highest levels of LiP and laccase are produced in the presence of dyes in the N-rich media. The presence of the dyes or varying N-conditions had no positive effect of stimulating higher MnP levels for P. Laccase had the highest activity. Lip activity was increased to 179. tremellosa. but the activity levels were still high in relation to the quantities of LiP produced by the other 3 fungi in Kirks media.7 3. although the enzyme activity was still quite high. 4. These findings are supported by work carried out by Kaal et al. / Enzyme and Microbial Technology 29 (2001) 575–579 degradation in the N-limited medium (Fig. Table 2 shows the enzyme activities of B. Laccase production began at an earlier stage under N-rich conditions. compared to after day 7. and their potential to produce similar quantities in the presence of dyes in both high and low nitrogen conditions.

[8] Tien M. Linkins AE. adusta successfully to degrade dyes in an artificial effluent. of Education. MnP. ostreatus. Method Enzym 1988. adusta and C. Enz Microb Technol 2001. 8:234 – 40. tremellosa. tremellosa in N-rich media. Bioremediation of textile industry waste water by white-rot fungi. Bioproc Eng 2000. Robinson et al. tremellosa and B. 579 References [1] Hatakka A. This study demonstrated the ability of the white-rot fungi tested to produce ligninolytic enzymes. [6] Kirby N. 5. Biores Technol 2000. Marchant R. tremellosa and had no positive effect on degradation by B.4⬘-dimethoxyphenyl)-2-(o-methoxyphenoxy)-propane-1. dye degradation. versicolor were all capable of producing LiP. Banat IM. adusta had lower enzyme activities than P. / Enzyme and Microbial Technology 29 (2001) 575–579 tion in N-rich media in relation to N-limited media. Method Enzym 1988. Lignin peroxidases of Phanerochaete chrysosporium. Joyce TW. Crawford RL. Biores Technol 1995. Feijoo G. Huynh V-B. 74% of the dyes were degraded under N-limited condition. Increasing ligninolytic activities in several white-rot basidomyces by nitrogen-sufficient media. Nigam P. degrading 79% of the dyes. . Lignin modifying enzymes from selected white-rot fungi: production and role in lignin degradation. [9] Paszczynski A. Lema JM. 161:264 –70. is thankful to the Dept. tremellosa yet degraded 85% of the dyes in N-rich media and 86% in N-limited media. LiP and laccase were produced in the highest activities. Conclusions P. Marchant R. Physical removal of textile dyes from effluents and solid-state fermentation of dye-adsorbed agricultural residues. Singh D. Production of phenol oxidases and peroxodases by wood rotting fungi. the amount of degradation was much the same. Northern Ireland (DENI) for a research studentship to carryout this work toward his PhD award. Zvavya R. Read JS.72: 219 –26. LiP activity doubled and laccase activity quandrupled for P. [4] Nigam P. Manganese peroxidase production by Bjerkandera sp BOS55. Mycol 1989. Armour G. Northern Ireland: University of Ulster. [2] Moreira MT. Although high nitrogen increased enzyme activities. Mswaka AY.R. Antibus RK. P. Acknowledgment T.28:420 – 6. [7] Reid ID.23:657– 61. 1999. [10] Szklarz GD. and the ability of P.13:125–35.70:453– 60. PhD Thesis. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Coleraine. adusta. and ligninolytic activity studies on Zimbabwean white-rot fungi. degradation was only increased slightly for P. [5] Robinson T.53:133–9.161:238 – 49. B. Growth. Dimethylation of lignin model dimer 1-(3⬘. [11] Kaal EEJ. Sinsabaugh RL.3-diol by the white-rot fungus Phlebia tremellosa.T. Can J Bot 1992. McMullan G. [3] Tekere M. Field JA. and laccase in Kirks media. B. Biores Technol 2001. FEMS Microbiol Rev 1994. Manganese peroxidase of Phanerochaete chrysosporium: purification. Kirk TK.77:247– 55.