Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

Enzyme and Microbial Technology 28 (2001) 420 – 426

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Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi M. Tekerea, A.Y. Mswakab, R. Zvauyaa, J.S. Reada,*,1 a

University of Zimbabwe, Department of Biochemistry, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe, Africa University of Zimbabwe, Department of Biological Sciences, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe, Africa

b

Received 22 June 2000; received in revised form 16 October 2000; accepted 9 November 2000

Abstract White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25–37°C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Bioremediation; Ligninolytic; Triphenylmethane and polymeric dyes

1. Introduction White rot fungi are a heterogenous group of organisms but have in common the capacity to degrade lignin as well as other wood components. The white rot fungi are by far the most efficient ligninolytic organisms described to date. This capability to degrade lignin is due to the extracellular nonspecific and nonstereoselective enzyme system. The extracellular enzyme system involved in lignin degradation is composed of lignin peroxidases, laccases and manganesedependent peroxidases as well as hydrogen producing oxidases [1–3]. The same unique nonspecific mechanisms that give these fungi the ability to degrade lignin also allow them to degrade a wide range of pollutants and they possess a number of advantages not associated with other bioremediation systems [4,5]. Because key components of the white * Corresponding author: Fax: ⫹263-9-286-803. 1 Present address: Department of Applied Biology and Biochemistry, National University of Science and Technology, P.O. Box AC939, Ascot, Bulawayo, Zimbabwe. E-mail address: [email protected] (J.S. Read).

rot lignin degrading system are extracellular, the fungi can degrade very insoluble chemicals such as lignin and many of the hazardous environmental pollutants. Furthermore they do not require pre-conditioning to a particular pollutant [1,3]. Researchers have focused mainly on Phanerochaete chrysosporium, however the possible practical application of this fungus does not always enable the optimum culture conditions to be fulfilled [2,6]. It may therefore be beneficial to screen a variety of white-rot fungi for their ability to degrade xenobiotics under a wide range of environmental conditions. In addition to screening established culture collection strains, new isolates can yield strains that are able to degrade xenobiotics more rapidly [2,7,8]. Tropical white rot fungi species which are widely represented in the hardwood forests of Zimbabwe, are the least studied with respect to their biodegradative capabilities. These tropical basidiomycetes contain several aggressive saprotrophs and they are tolerant to harsh tropical environmental conditions [9]. Dye decolorization has been proposed as a quick, reproducible and inexpensive screening method to determine ligninolytic activity and the ability to degrade aromatic compounds

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M. Tekere et al. / Enzyme and Microbial Technology 28 (2001) 420 – 426

[2,10]. In this study the polymeric dyes; Poly R478 and blue dextran were used together with the triphenylmethane dyes; crystal violet, bromophenol blue and cresol red for screening and degradative studies. Triphenylmethane dyes have been reported to undergo extensive biodegradation in ligninolytic cultures of P. chrysosporium [2,11]. Triphenylmethane dyes have found extensive use in human and veterinary medicine as biological stains and as textile dyes. Unfortunately waste water treatment facilities are often unable to completely remove commercial dyestuffs from contaminated waste water, and contribute to the pollution of the aqueous environment [11]. There are possibilities therefore for the use of the white rot fungi for treating factory wastes. Locally collected species were studied with reference to their optimum growth temperatures, dye decolorization, hydrolytic and ligninolytic enzyme activities. A simple plate test for direct visualization of biological lignin degradation was done to evaluate the presence of ligninolytic activity in the cultures [12], to compare the dye decolorization screening with ligninolytic activities and related enzyme activities. Semiquantitative determination of hydrolytic enzyme activities using API ZYM system was done on the cultures to characterize their more general degradative capabilities.

2. Materials and methods 2.1. Chemicals All the chemicals used were reagent grade unless otherwise stated. Poly R478, blue dextran, crystal violet, cresol red and bromophenol blue were obtained from Sigma Chemical Co., St. Louis, MO. Trivial names are used here for the dyes as a matter of convenience. Alkali lignin was obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis. 2.2. Organisms The following isolates were used, Trametes elegans (Berk.) Ryvarden TEL152, Trametes versicolor (Fr.) Pilat TVE123, Trametes cingulata (Berk.) Ryvarden, Trametes pocas (Berk.) Ryvarden TPO119, Isolate DSPM95, Datronia concentrica (Berk.) Ryvarden, Irpex spp* IST144, Lentinus velutinus, (Fr) Creptidotus mollis (Schaeff.:Fr.) Kummer and Pycnoporus sanguineus (Fr) Murill. The fungal strains were collected in August, 1995 from Chirinda, a moist evergreen forest, and Chimanimani (miombo woodland) forests of Zimbabwe. The fungi were growing on dead wood. These isolates were dominant in these forests at the time of collection and a wider survey on the distribution of the white rot fungi done by Mswaka [13] show that these fungi are common in most Zimbabwean woodlands. To obtain pure cultures, small fragments about 1 mm diameter from the inner flesh of the basidiocarp were plated onto 3% malt extract agar (MEA) plates and mycelium were repeatedly transferred onto new plates until the cultures were pure.

421

Stock cultures were stored on MEA plates at 4°C and periodically subcultured. With the exception of Irpex spp* IST144 and DSPM95, all the other isolates used were identified using gross and microscopic characteristics [13]. They are all deposited with the Department of Biological Sciences culture collection, University of Zimbabwe. 2.3. Effect of temperature on the growth of the isolates 6 mm agar plugs from the growing margin of test strains were inoculated onto 3% malt agar plates. Replicates were prepared for each isolate and incubated at each of the following temperatures; 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C and 45°C. The experiment was repeated twice. Growth was followed by measuring radial extension of the mycelium every 24 h for at least a week. Mean growth rates (mm/day) were calculated, then these were combined to give the optimum growth temperature. 2.4. Dye decolorization experiments The medium used consisted of (w/v), 2% glucose, 3% malt extract, 0.02% dye, 0,1% mycological peptone. 1% agar was used for dye decolorization tests on plates. A 6 mm agar plug from the growing margin of test strains were used as inoculum. Plates were prepared in triplicate for each isolate and incubated at 30°C for isolates with a high growth temperature optimum and 25°C for those with temperature optimum below 30°C. The extent of decolorization was assessed by visual examination of the decolorization area and qualitatively determined by assigning numbers 0 –5 with decolorization maximum at 5. The isolates were later tested for Poly R478 degradation in shake flasks. Four 6 mm agar plugs were used to inoculate 100 ml of the medium above in 500 ml Erlenmeyer flasks and the flasks were incubated on a shaker at 100 rpm. The decolorization was determined by measuring the absorbance ratio 520/350 nm using a Shimadzu Biospec-1601 [10]. Duplicate flasks were used for each isolate of the experiments was done at least twice. The effect of initial pH on Poly R478 decolorization by those isolates showing high decolorization was also studied so as to determine the optimum initial pH of decolorization for the fungi and its variability from one isolate to the other. A citrate - phosphate buffer system consisting of 0.1 M citric acid and 0.2 M Na2HPO4. 7H20, pH 2.6 to pH 6.0 was used. 2.5. Plate assay for lignin biodegradation The plate assay method by Sundman and Nase [12] was adopted. The medium used had the following composition; 5 g glucose, 5 g ammonium tartrate, 1 g malt extract, 0.5 g MgSO4.7H2O, 0.01 g CaCl2. 2H2O, 0.1 g NaCl, 0.01 g FeCl3, 1 mg thiamine and 20 g agar in a liter of distilled water with pH adjusted to 4.5. Alkaline lignin (0.025%) was

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M. Tekere et al. / Enzyme and Microbial Technology 28 (2001) 420 – 426

used in the medium. The test agar plates were inoculated with a 6 mm agar plug from a growing plate and one isolate was used on each plate. Three replicate plates were used for each isolate and the experiment was repeated twice. Cultures were incubated at 30°C for 10 days after which the mycelia was scraped off the plates which were then flooded with a reagent containing equal parts of 1% aqueous solutions of FeCl3 and K3[Fe(CN)6]. A positive result was indicated by clear zones under or around the growth area of lignin-degrading fungi. 2.6. Lignin peroxidase, manganese peroxidase and laccase activity Ligninases were produced in shallow stationary cultures using the basal medium proposed by Bonnarme et al. [14] 4 ⫻ 6 mm agar plugs from 7 day old cultures were used for inoculating 50 ml sterile medium in 500 ml Erlenmeyer flask and incubated at 30°C in the dark. The medium was sterilized by autoclaving at 125°C for 15 min except for thiamine which was filter sterilized. Centrifuged extracellular fluids (10 000 rpm for 10 min) were utilized for enzyme assays. All enzymes were determined spectrophotometrically at room temperature using a Shimadzu Biospec-1601. Although the experiments were repeated, mean results for duplicates from one set of experiments are reported because of the different activity levels obtained in different sets of experiments but the trend of the activities was however always the same. Lignin peroxidase activity was determined using the method of Tien and Kirk (1984) [15]. To verify the lignin peroxidase assay method, P. chrysoporium BKM-F-1767 ATCC 24725 (obtained from de Bont, J.A.M., Division of Industrial Microbiology, Dept. of Food Science, Wageningen University, Netherlands) was used alongside as a reference strain. Up to 0.6 ml culture supernatant was used in the assay. Manganese (II) peroxidase activity was determined by following the formation of a complex between Mn3⫹ and lactate at 300 nm [16,17]. The reaction mixture contained 0.5 ml of 0.01 M Mn2⫹, in 100 mM sodium lactate buffer pH 4.5, 0.1 mM H2O2 and 0.5 ml culture supernatant. Laccase activity was determined using 2,6-dimethoxyphenol (DMP) as substrate at 468 nm. The reaction mixture contained 50 mM sodium malonate (pH 4.5), 1 mM DMP and 0.25 ml enzyme supernatant [10]. 2.7. Enzymatic activities determination using the API ZYM system The fungal isolates were grown on 0.5% mycological peptone and 3% MEA plates in duplicate and the experiment was repeated twice. Mycelia from a five day growth on a plate was used to prepare a suspension. All the other experimental procedures were as outlined in the API ZYM protocol supplied by bioMerieux, Marcy-l‘Etoile, France.

Table 1 Decolorisation magnitudes for the dyes crystal violet, bromophenol blue, cresol red, blue dextran, and ligninolysis by the fungal isolates Isolate

Lignin C. violet Dye B. dextran degradation B. blue C. red

Trametes elegans Trametes cingulata Trametes pocas Trametes versicolor DSPM95 Datronica concentrica Irpex spp Lentinus velutinus Creptidotus mollis Pycnoporus sanguineus

⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹ ⫹

1 2 4 5 5 2 1 3 3 2

3 5 3 5 5 5 5 4 1 3

3 4 5 5 5 3 5 2 3 3

3 5 2 5 5 3 3 1 1 2

C. violet - crystal violet, B. blue - bromophenol blue, C. red - cresol red, B. dextran - blue dextran, and (⫹), (⫹⫹), (⫹⫹⫹) - increasing ligninolysis magnitude.

Incubation of the strips was done at 25°C. The color reactions were assigned values ranging from 0 –5 as per the color chart supplied by the manufacturer. Values 1 through to 4 were intermediate reactions depending on the level of intensity.

3. Results 3.1. Effect of temperature on growth of the isolates The determined optimum growth temperatures for these fungi isolated from cool high rainfall semi-tropical forests ranged from 25 to 37°C. The optimum growth temperatures obtained for the fungal isolates were: T. pocas, 30 –35°C; T. versicolor, 30°C; T. cingulata; 37°C; D. concentrica; 35°C; Isolate DSPM95, 35– 40°C; C. mollis, 25°C; P. sanguineus, 37– 40°C; L. velutinus, 30°C; Irpex spp, 30°C and T. elegans, 25°C. Optimum growth rates (mm/day) as determined from optimum growth temperatures were: T. pocas, 10.0 ⫾ 0.7; T. versicolor, 7.0 ⫾ 0.5; T. cingulata, 10.0 ⫾ 0.4; D. concentrica, 11.0 ⫾ 0.3; Isolate DSPM95, 12,.5 ⫾ 0.8; C. mollis, 4.0 ⫾ 0.4; P. sanguineus, 11.5 ⫾ 0.7; L. velutinus, 9.0 ⫾ 0.2; Irpex spp, 9.5 ⫾ 0.8 and T. elegans, 4.0 ⫾ 0.4. 3.2. Lignin degradation and dye decolorization studies All the isolates showed some lignin degradative activity, with D. concentrica, T. versicolor and DSPM95 showing the highest magnitudes of ligninolysis. The results on the lignin degradation are shown in Table 1 and it is apparent that lignin degradation in D. concentrica is not reflected in an uniformally high dye degradative abilities. An opposite result is shown with T. cingulata in particular, as a fairly good dye degradation on agar plates was not matched with ligninolysis on the lignin agar plates.

M. Tekere et al. / Enzyme and Microbial Technology 28 (2001) 420 – 426

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Table 2 Changes in initial pH during the decolorisation of Poly R478 by the fungal isolates in medium buffered with a citrate-phosphate buffer system consisting of 0.1 M citric acid and 0.2 M Na2HPO4. 7H2O, pH 2.6 to pH 6.0. Initial pH was measured at the beginning of the culturing and final pH measured at the end of culturing to determine the changes in pH during the decolorisation of Poly R478 by the fungal isolates Isolate

DSPM95 T. versicolor T. pocas T. cingulata P. sanguineus D. concentrica

Initial pH 4.5

2.5

3

4

2.70 2.70 2.67 2.64 2.67 2.70

3.45 3.13 3.25 3.26 3.21 3.57

Final 4.16 4.18 4.15 4.11 3.88 4.04

pH 4.05 4.64 4.41 4.45 4.50 3.95

5

6

4.56 4.96 4.6 4.81 4.48 4.54

4.96 4.25 4.23 5.10 4.46 3.88

3.3. Effect of initial pH on Poly R478 decolorization

Fig. 1. (A) Poly R478 decolorization by white rot isolates in liquid cultures as a function of time as determined by absorbance ratio A520/350 nm. T. pocas, T. versicolor, T. cingulata, D. concentrica, Isolate DSPM95 and ⴛ P. sanguineus. The error bars represent the calculated standard deviation of the samples. (B) Poly R478 decolorization by white rot isolates in liquid cultures as a function of time as determined by absorbance ratio A520/350 nm. 䉬 C. mollis, L. velutinus,  Irpex spp and 䉫 T. elegans. The error bars represent the calculated standard deviation of the samples.

The decolorization magnitude (scale 1–5) of the different dyes by the fungal isolates on agar plates is shown in Table 1. Fig. 1(A) and 1(B) shows the results of decolorization of Poly R478 by the isolates in liquid cultures. Isolate DSPM95, T. pocas, T. cingulata and T. versicolor showed extensive decolorization both on agar plates and the liquid culture decolorization of Poly R478 and in both cases isolate DSPM95 and T. versicolor degraded the dyes most. Irpex spp, P. sanguineus and T. elegans showed some significant decolorization capabilities on agar plates for bromophenol blue, blue dextran and cresol red but only minimum activities were observed in liquid cultures for Poly R478 decolorization as shown in Fig. 1(B). With the exception of D. concentrica all the other isolates shown in Fig. 1(A) had decolorization activities in liquid media which were almost equal to T. versicolor, which is one of the isolates known to have extensive ligninolytic activities [6,18 –20].

The optimum pH for Poly R478 decolorization was generally low, between 3.0 and 5.0. Isolates T. pocas and P. sanguineus had an optimum pH of 4.5 for Poly R478 decolorization. T. versicolor and DSPM95 showed high decolorization when the pH was between 4.5 and 6.0 with maximum degradation at pH 5.0. With T. cingulata there was a much narrow pH tolerance with an optimum pH of 3.0. There was a broad pH tolerance with the isolate D. concentrica, giving almost the same percentage of decolorization from pH 3.0 to pH 6.0. Measurement of the final pH at the end of experiment (Table 2) showed small but consistent upward movement from low pH toward the optimum pH and similarly, a decrease from higher pH to lower pH for all isolates. 3.4. Hydrolytic enzyme activities of the white rot fungal species using API ZYM system The hydrolytic enzyme activities for all isolates are shown in Table 3. All the isolates showed no activity for the enzymes; trypsin, ␣-mannosidase, ␣-fucosidase and the lipase (C14). Most isolates displayed extensive esterase (C8), carbohydrase, acid phosphatase and leucine arylamidase activity as well as cellulosic complex enzyme activity. The enzyme activities were low for most isolates for the following enzymes; alkaline phosphatase, esterase (C4), valine arylamidase and cystine arylamidase. 3.5. Lignin peroxidase, manganese peroxidase and laccase activity All strains had manganese peroxidase and laccase activities. For manganese peroxidase and laccase activities, results are shown in Figs. 2 and 3 respectively. No lignin peroxidase activity was detected in all the isolates studied except in the reference strain, P. chrysosporium (results not shown). Even when Mn2⫹ which is known to reduce LiP

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Table 3 Hydrolytic enzyme activities in white rot fungi isolates as determined by the API ZYM reactions Enzyme assayed for

P. sanguineus C. mollis Irpex spp T. elegans T. versicolor T. pocas T. cingulata DSPM95 L. velutinus D. concentrica

Control Phosphate alkaline Esterase (C4) Esterase (C8) Lipase (C14) Leucine arylamidase Valine arylmidase Cystine arylamidase Trypsin Chymotrypsin Acid phosphatase Napthol-AS-BIphosphohydrolase ␣-galactosidase ␤-galactosidase ␤-glucuronidase ␣-glucosidase ␤-glucosidase N-acetyl-␤glucosaminidase ␣-mannosidase ␣-fucosidase

0 0 0 1 0 1 2 1 0 0 1 2

0 1 0 1 0 4 1 1 0 0 5 1

0 1 0 2 0 1 1 0 0 0 2 5

0 3 1 2 0 5 1 1 0 1 5 5

0 1 1 2 0 3 1 0 0 0 5 3

0 1 0 1 0 5 1 0 0 0 0 5

0 1 1 3 0 5 3 3 0 0 5 5

0 0 0 1 0 5 0 0 0 0 1 3

0 0 1 1 0 2 0 0 0 0 5 0

0 0 1 0 0 1 0 0 0 0 5 1

1 1 5 3 3 5

1 1 0 0 2 0

2 2 5 2 0 5

2 0 5 1 2 1

1 3 3 2 5 1

1 2 1 1 0 1

3 0 3 0 5 5

0 1 5 2 1 3

1 0 0 0 1 1

2 1 1 0 5 1

0 1

0 0

0 0

0 0

1 0

0 0

0 0

0 0

0 0

0 0

titers [14], was excluded or when veratryl alcohol was added as an inducer, no LiP was detectable under these culture conditions. The manganese peroxidase and laccase activities were obtained after at least 3 days of culture growth. Manganese peroxidase levels were variable within the isolates, high activities were observed in T. versicolor, isolate DSPM95, T. cingulata and T. pocas cultures. Laccase activity was highest in DSPM95 followed by T. versicolor, L. velutinus and lowest activity was in T. elegans.

4. Discussion The white rot fungi studied have variable growth temperature optima, including those from the Trametes genus despite the fact that they were isolated from the same macro-environment. A range of temperature optima amongst the isolates may mean that a specific isolate can be selected for particular conditions. The fast growing fungi were usually those with a high temperature optima. All the isolates studied showed some dye decolorization demonstrating the potential bioremediation capabilities of the isolates. Comparison of the isolates with the isolate T. versicolor, one of the more extensively studied species after P. chrysosporium [6,19,20], shows that the isolates DSPM95, T. pocas, T. cingulata and P. sanguineus, are equally competitive for use in biodegradation based on the magnitude of the decolorization of the dyes obtained. The study of the effect of pH changes on the degradation of Poly R478 was interesting as some of the isolates were

able to maintain the degradation over a wide range of pH, up to pH 6.0 (T. versicolor, D. concentrica and DSPM95). The other group (T. cingulata, T. pocas and P. sanguineus) however showed a distinct preference for acid conditions for degradation, with degradation declining markedly after pH 4.5. This suggests an interesting area for further investigation, it could be possible that this reflects the enzymic optimum range, but it can also be the effect of pH on the fungal metabolism and spectrum of enzymes produced by each isolate. The determined final pH of the medium show that there was obviously a variable response to the way each isolate was able to affect the buffered medium. The observed lowering of pH could be attributed to de novo organic acids production during secondary metabolism [3]. From the results of the pH studies, pH 3.0 is suitable for biodegradative studies with isolate T. cingulata while pH 4.5 can be used for biodegradative studies with the isolates: T. pocas, P. sanguineus, D. concentrica, T. versicolor and DSPM95 since it is within their optimum pH range for decolorization. The wide host range and tolerance of harsh sub-tropical environmental conditions is indicative of a highly adaptable physiology and heterogenous enzyme system in these white rot fungi [9]. The API ZYM system confirmed the diversity of the hydrolytic enzymatic activities that are present in these white rot fungi. These fungal isolates can also be considered as potential sources of other degradative enzymes in particular those that hydrolyze different carbohydrate compounds. Mswaka and Magan [9] reported extensive and variable endo-glucanase and exo-glucanase activities in the tropical basidiomycetes they studied.

M. Tekere et al. / Enzyme and Microbial Technology 28 (2001) 420 – 426

Fig. 2. (A) MnP activity in white rot fungi isolates. T. pocas, T. versicolor, T. cingulata, D. concentrica, Isolate DSPM95, ⴛ P. sanguineus. (B) MnP activity in white rot fungi isolates. 䉬 C. mollis, L. velutinus, 䉫 T. elegans and  Irpex spp.

The presence of MnP and laccase activities agreed with the findings obtained by other researchers that MnP-laccase combination is the most common group of enzymes in the white rot fungi [21,22]. It is also proposed that the activity of MnP and/or laccase may be sufficient for lignin degradation in some fungi [21]. Our negative lignin peroxidase results may suggest that these fungi either produce no significant levels of this enzyme [3] or its production requires different conditions [22]. The latter was true in previous studies on Trametes versicolor and Bjerkandera adusta which are known as lignin peroxidase producers [6,23]. The study of ligninolytic enzymes in other white rot fungi also concluded that lignin peroxidase production is highly dependent on the culture conditions and/or is strain related [21]. During our studies we tested for lignin peroxidase production under varied concentrations of carbon, nitrogen and manganese and still this did not yield lignin peroxidase activity. Comparing the MnP and laccase activities with ligninolysis on plates and the dye decolorization, high ligninolytic ability in DSPM95 and T. versicolor can be easily

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Fig. 3. (A) Laccase activity in white rot fungi isolates. T. pocas, T. versicolor, Isolate DSPM95, L. velutinus, D. concentrica and  Irpex spp. (B) Laccase activity in white rot fungi isolates T. cingulata, 䉬 C. mollis, ⴛ P. sanguineus and 䉫 T. elegans.

correlated with high dye decolorization. However in the case of D. concentrica where there was high ligninolysis on plates but no detectable lignin peroxidase, and low manganese peroxidase and laccase activity present, it is difficult to correlate these facts. The residual activity could be due to other mechanisms as proposed by Bumpus and Brock, (1988) [11]. These authors observed an 89% decolorization of crystal violet in non-ligninolytic cultures of P. chrysosporium, which they attributed to other degradative mechanisms rather than the ligninolytic system. However the fact that dye decolorization (Table 1) does not always match the ligninolytic results should not invalidate the dye screening methods as dye degradation may be a useful property for further studies [2]. Our results also show that plate screening should be complimented by liquid culture screening. Isolate DSPM95 shows high manganese peroxidase and laccase activities, high dye decolorization rates and has fast growth rates, this isolate has been selected for further studies.

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Acknowledgments The authors acknowledge the Swedish Agency for Research Cooperation with Developing Countries (SAREC) for the financial support of this study.

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