Studies on the production of enzymes by white-rot fungi for thedecolourisation 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 abilityto produce Lignin Peroxidase (LiP), Manganese Peroxidase (MnP), and Laccase in a nitrogen deficient mineral salts medium. B. adusta andP. 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 100mg/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 degradedin 9 days by B. adusta and 74% by P. tremellosa in 11 days in N-limited conditions. Addition of nitrogen had no substantial effect onpercentage dye degradation by B. adusta, with a slight increase for P. tremellosa. Nitrogen supplementation did however reduce the decolouri-sation time. The results show the potential of using B. adusta and P. tremellosa for textile- effluent degradation, with or without nitrogensupplementation.

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 thelignin- degrading capabilities of white-rot fungi [1]. Thesenon-specific enzymes which allow white-rot fungi to de-grade lignin, also allows them to degrade a wide range ofpollutants. This has led to research regarding their use forthe degradation of recalcitrant compounds, such as, polycy-clic aromatic hydrocarbons (PAH’s) and dyes [2,3].

There are more than 100,000 commercial dyes availableto textile industries who consume vast quantities of waterand chemicals during processing, ultimately producing dye-containing effluent which consists of complex, syntheticand often recalcitrant compounds. Dyes are designed to beresistant to light, water and oxidising agents and are there-fore difficult to degrade once released into aquatic systems[4,5]. Conventional treatments of textile effluents are eitherineffective, costly, complicated or have sludge disposal

problems, so the potential exists for the breakdown of dyesin effluent by extracellular enzymes produced by white-rotfungi during fermentation.

This study assessed the enzyme activities of four white-rot fungi in a nitrogen deficient mineral salts medium. Fromthis two were selected for their potential to degrade 5 dyesin an artificial textile- effluent in both N-rich and N-limitedconditions.

2. Materials and methods

2.1. Chemicals

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

* 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).

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2.2. White-rot fungi

The following white-rot fungi were used, Bjerkanderaadusta, Phlebia tremellosa, Pleurotus ostreatus, Coriolousversicolor. All cultures were obtained from the CanadianCulture Collection, Alberta, Canada. Fungi were maintainedon PDA plates and stored at 4°C and subcultured everymonth.

2.3. Enzyme activity experiments

10 mm plugs were taken from stock plates and wereinoculated into 50 ml of Malt Extract Broth. This wascarried out in duplicate for each fungus and incubated stat-ically at optimal temperatures for each culture for 5 days.After 5 days excess media was drained and the fungalmycelia macerated.

5 ml of the homogenised mycelia was then transferred to45 ml of Kirks media (and P. tremellosa media). The fungiwere again incubated statically at the various optimal tem-peratures. Samples were taken at regular intervals, centri-fuged, and the supernatant used to measure enzyme activi-ties in a spectrophotometer. From this, two fungi wereselected for their ability to produce enzymes and decolour-ise an artificial textile-effluent consisting of 5 dyes.

2.4. Media compositions

The media used for measurement of enzyme activity wasa nitrogen deficient mineral salts Kirks medium, as de-scribed by Kirby [6]. It had the following composition:KH2PO4 (0.20 g/l), MgSO4 � 7H2O (0.05 g/l), CaCl2 (0.01g/l), ammonium tartrate (0.22 g/l), 2,2-dimethylsuccinicacid (2.90 g/l). The following were added post autoclaving:glucose (10 g/l), thiamine (0.10 g/l), trace elements (10.0ml/liter), Tween 80 (0.10% v/v), veratryl alcohol (1.5 mM).The trace elements solution contained: CuSO4 � 7H2O (80mg/liter), H2MoO4 (50 mg/liter), MnSO4 (33 mg/liter),ZnSO4 � 7H2O (43 mg/liter) and Fe(SO4)3 (50 mg/liter) andwas filter sterilised prior to use.

The mineral salts media used for P. tremellosa wasdescribed by Reid [7]. Kirks media, as described above, wasalso used for dye decolourisation experiments. Similar en-zyme activities were found for P. tremellosa grown on thespecialised media and Kirks media, so Kirks was used forthe decolourisation experiments.

Dye decolourisation experiments were carried out inN-rich and N-limiting conditions, with 0.2% peptone addedto produce a C:N ratio of 11.6:1 compared to 116:1 forN-rich media.

2.5. Dye decolourisation experiments

5.ml of homogenised mycelium from each fungus wereadded, with 5 ml of 1000 mg/liter of artificial effluent stocksolution, to 40 ml of Kirks media. Experiments were carriedout in duplicate for both N-rich and N-limiting conditions.

2.6. Enzyme assays

All enzymes were determined spectrophotomerically.LiP activity was performed according to the method of Tienand Kirk [8]. MnP activity was measured by the method ofPaszczynski et al. [9]. Laccase activity was determined bythe method of Szklarz et al. [10]. One unit of enzymeactivity was defined as 1 �M of veratryl alcohol or quinoneoxidised in 1 min under defined conditions, and activitiesreported as UL�1.

3. Results

3.1. LiP, MnP and laccase activities

All fungi had LiP, MnP and laccase activity, with MnPbeing produced in the smallest quantities. Enzyme activitiesgenerally began between days 1 and 3, with peak activitiesbetween days 6 and 9. P. tremellosa and B. adusta had thehighest MnP activities of the 4 white-rot fungi, with P.tremellosa having the highest laccase activity. B. adusta hadthe highest production of LiP, but the lowest of the 4regarding laccase activities. P. ostreatus and C. versicolorproduced 100 and 82 UL�1 of LiP respectively, but onlysmall amounts of MnP (Table 1).

3.2. Decolourisation of textile-effluent in N-rich medium

B. adusta produced a decrease in the textile-dye effluentwith a corresponding increase in enzyme activity over theperiod of fermentation, with LiP activity peaking at the timeof maximal degradation (Fig. 1a). Laccase activity fluctu-ated, although activity increased during periods of dye deg-

Table 1Peak enzyme activities on N-limiting Kirk’s medium (without artificial effluent)

B. adusta P. tremellosa P. ostreatus C. versicolor

Peak UL�1 Day Peak UL�1 Day Peak UL�1 Day Peak UL�1 Day

LiP 170.3 6 98.2 6 101.9 9 83.4 8MnP 5.1 9 3.9 12 1.58 9 0.59 6Laccase 20.8 8 98.4 8 38.6 4 32.1 11

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radation. A total of 85% of the dyes in the artificial effluentwere degraded and a period of 7 days was required for theachieved decolourisation through degradation of the dyecompounds.

P. tremellosa successfully degraded 79% of the artificialeffluent after 9 days. There were sharp increases in LiP andMnP activities between days 7 and 9, corresponding to adecline in the dye concentrations left in the effluent (Fig. 1b).

3.3. Decolourisation of textile-effluent in N-limitedmedium

In N-limited conditions B. adusta degraded a similaramount of textile-effluent in N-rich media (Fig. 2a). Al-

though the amount of dye degraded was similar, the amountof enzyme activity under N-rich conditions was muchhigher. A lag phase of degradation occurred at the beginningof the fermentation in both nitrogen levels, but a longer lagphase was recorded in the N-limiting conditions, from day1–5 for both fungi after which most of the dyes weredegraded. Higher activity levels of LiP were maintained forboth organisms throughout the fermentation period in N-limited conditions, with a high initial production after day 1.

P. tremellosa degraded 74% of the artificial-effluent in11 days in N-limited conditions, with a slightly higherdegree of degradation. B. adusta degraded 85% of the dyesin N-rich conditions in 9 days with a similar amount of

Fig. 1. (A) Production of lignolytic enzymes and textile-effluent decolourisation in N-rich media by B. adusta. —■ — effluent decolourisation (%), --�--MnP, --Œ-- laccase, --�--, LiP. (B) Production of lignolytic enzymes and textile-effluent decolourisation in N-rich media by P. tremellosa. —■ — effluentdecolourisation (%), --�-- MnP, --Œ-- laccase, --�-- LiP.

Fig. 2. (A) Production o f lignolytic enzymes and textile-effluent decolourisation in N-limited media by B. adusta. —■ — effluent decolourisation (%), --�--MnP, --Œ-- laccase, --�-- LiP. (B) Production of lignolytic enzymes and textile-effluent decolourisation in N-limited media by P. tremellosa. —■ — effluentdecolourisation (%), --�-- MnP, --Œ-- laccase, --�-- LiP.

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degradation in the N-limited medium (Fig. 2b). Laccaseproduction began at an earlier stage under N-rich condi-tions, after day 3, compared to after day 7, N-rich media.

3.4. Effect of dyes and nitrogen content on enzymeactivities

P. tremellosa and B. adusta were grown in the presenceof 100 mg/liter of the artificial effluent at both high and lowN levels. Lip activity was increased to 179.9 UL�1 in N-richmedia, with a smaller increase in N-limited conditions,114.7 UL�1. The presence of the dyes or varying N-condi-tions had no positive effect of stimulating higher MnP levelsfor P. tremellosa, although laccase levels were increased 4fold in the presence of the artificial effluent in N-rich media.In N-limited conditions laccase activity was doubled.

B. adusta showed a slight decrease in LiP productionwhen grown in dye and both nitrogen varying media, al-though the enzyme activity was still quite high. MnP activ-ity was reduced by a quarter in both nitrogen conditions. Anincrease in laccase activity was noted in N-rich conditions,with a slight fall in activity when grown in N-limited media.

4. Discussion

P. tremellosa and B. adusta were selected for their highenzyme activities, and their potential to produce similarquantities in the presence of dyes in both high and lownitrogen conditions. P. ostreatus and C. versicolor did notproduce the same high enyme activities for LiP and laccaseand so were not used for the textile-effluent degradationexperiments.

The main enzymes that appear to be used for dye deg-radation are LiP and laccase, as they were produced in thehighest quantities with and without the presence of dyes andat the two different nitrogen conditions. The amount of LiPincreased in the presence of the artificial textile-effluent inboth nitrogen conditions for P. tremellosa. The LiP activitydid not increase as dramatically in the presence of the dyesin B. adusta. There was in fact a small decline in LiPactivity, but the activity levels were still high in relation tothe quantities of LiP produced by the other 3 fungi in Kirksmedia. Laccase activity by P. tremellosa increased dramat-ically (44%) in N-rich media, suggesting that the presenceof dyes and the nitrogen supplement led to an increase inlaccase production. Table 2 shows the enzyme activities ofB. adusta and P. tremellosa in the presence of dyes and atthe different nitrogen conditions.

The results concerning P. tremellosa, seem to indicatethat laccase is one of the major ligninolytic enzymes in-volved in the breakdown of the dyes in the artificial effluent.Laccase had the highest activity. LiP and laccase showedthe biggest increase of the 3 enzymes during the degradation

of the effluent. The highest levels of LiP and laccase areproduced in the presence of dyes in the N-rich media, withalmost twice as much laccase being produced in N-richconditions compared to that produced in N-limited condi-tions (Table 2). These findings are supported by work car-ried out by Kaal et al., [11]. They found that higher ligni-nolytic activities were recorded in an enriched nitrogenmedium. Although the percentage decolourisation is 5%higher in the N-rich media, the higher enzyme activitiesseem to have had no substantial effect on degrading any ofthe dye effluent further. Whether or not the additional ni-trogen should be added to increase degradation slightly isvery much dependant on cost.

Kirby [6], showed that P. tremellosa was capable ofdecolourising a different artificial effluent, 70% degradationover a period of 14 days. This study produced a betterpercentage decolourisation through dye degradation in ashorter fermentation time with higher enzyme activities.

In this study degradation in both N-rich and N-limitedconditions occurred gradually over a period of 9 days.Degradation by B. adusta occured rapidly over a period ofdays in both N-rich and N-limited conditions, with most dyebreakdown occurring between days 3–7 in N-rich media andbetween days 5–9 in N-limited media. N-rich media had theeffect of causing dye degradation to begin earlier.

The major enzymes involved in dye degradation by B.adusta are also LiP and laccase. Assistance in the decolouri-sation of the effluent may be credited to other enzymespresent in the culture supernatant and mycelia bound en-zymes, which were not assayed for. Although the amountsof MnP were low, using the current assay, it cannot beconcluded if MnP played any role in aiding dye decolouri-sation. Similarly to P. tremellosa, higher enzyme activitieswere observed in N-rich media compared to N-limited me-dia, although the percentage of dye degradation was verysimilar in both nitrogen conditions. This also agrees with theenzyme activities of P. tremellosa at the two nitrogen con-ditions. Although there was an increase in enzyme produc-

Table 2Effect of nitrogen conditions on enzyme synthesis and textile-effluentdecolourisation

Fungi and enzymes N-richfermentation(UL�1)

N-limitedfermentation(UL�1)

P. tremellosaLiP 179.9 114.7MnP 3.93 3.4Laccase 390.6 218.5% decolourisation 79.1% 73.9%

B. adustaLiP 132.9 112.74MnP 0.9 0.72Laccase 22.6 13.5% decolourisation 84.9% 85.7%

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tion in N-rich media in relation to N-limited media, theamount of degradation was much the same.

5. Conclusions

P. ostreatus, P. tremellosa, B. adusta and C. versicolorwere all capable of producing LiP, MnP, and laccase inKirks media. LiP and laccase were produced in the highestactivities. LiP activity doubled and laccase activity quan-drupled for P. tremellosa in N-rich media, degrading 79%of the dyes. 74% of the dyes were degraded under N-limitedcondition. B. adusta had lower enzyme activities than P.tremellosa yet degraded 85% of the dyes in N-rich mediaand 86% in N-limited media.

This study demonstrated the ability of the white-rot fungitested to produce ligninolytic enzymes, and the ability of P.tremellosa and B. adusta successfully to degrade dyes in anartificial effluent. Although high nitrogen increased enzymeactivities, degradation was only increased slightly for P. tre-mellosa and had no positive effect on degradation by B. adusta.

Acknowledgment

T.R. is thankful to the Dept. of Education, NorthernIreland (DENI) for a research studentship to carryout thiswork toward his PhD award.

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