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Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461
Process Biochemistry 41 (2006) 941–946 www.elsevier.com/locate/procbio
Short communication
Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461 Ivana Eichlerova´ *, Ladislav Homolka, Frantisˇek Nerud Institute of Microbiology AS CR, Vı´deo`ska´ 1083, 142 20 Prague 4, Czech Republic Received 9 June 2005; received in revised form 14 September 2005; accepted 13 October 2005
Abstract Eight different Pleurotus species were tested for their Orange G and Remazol Brilliant Blue R (RBBR) decolorization capacity and their ligninolytic properties. All the species produced laccase (Lac) and manganese peroxidase (MnP) and in five species we found aryl-alcohol oxidase (AAO) activity. Strain CCBAS 461 of a very little studied species Pleurotus calyptratus was chosen for a more detailed study. This strain produced a relatively high amount of Lac, MnP and also AAO. Within 14 days the strain decolorized up to 91% of Orange G and 85% of RBBR in liquid culture and more than 50% of these dyes on agar plates. P. calyptratus is able to decolorize efficiently also other azo and phthalocyanine dyes, but only a limited decolorization capacity was found in the case of polyaromatic and triphenylmethane dyes. Lac and MnP production was strongly influenced by the kind of cultivation media and by the dye present. # 2005 Elsevier Ltd. All rights reserved. Keywords: Decolorization; Synthetic dyes; Pleurotus calyptratus; Laccase; Manganese peroxidase; Aryl-alcohol oxidase
1. Introduction Dye wastewater from textile and dyestuff industries is very difficult to treat. Synthetic dyes, classified by their chromophore as azo, anthraquinone, triphenylmethane, heterocyclic or phthalocyanine dyes, are very stable and resistant to microbial attack and therefore it is difficult to remove them from effluents by conventional biological processes. Decolorization of industrial dyes can be achieved by physico-chemical methods, such as adsorption, precipitation or chemical degradation, but these are very expensive, which limits their application [1]. The decolorization of the dyes through intermediates of different color has been proposed to be a series of multiple reactions. In case of anaerobic decolorization of azo dyes by some bacteria, arising intermediates and final products are carcinogenic and mostly more toxic than the starting dyes. In recent years, the utilization of biodegradative abilities of some white rot fungi seems to be promising [2]. Owing to their extracellular nonspecific free radical-based enzymatic system they can completely eliminate a variety of xenobiotics, including synthetic dyes, giving rise to non-toxic compounds [3,4].
* Corresponding author. Tel.: +42 241062611; fax: +42 241062384. E-mail address: [email protected] (I. Eichlerova´). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.018
Many white rot fungi (e.g. Phanerochaete chrysosporium, Bjerkandera adusta, Trametes versicolor etc.) have been intensively studied in connection with the decolorization processes [5–7]. Many papers described also biodegradative and decolorization abilities of several species of the genus Pleurotus, especially P. ostreatus [8,9], P. pulmonarius [10,11], P. eryngii [12] or P. sajor-caju [13]. Nevertheless, no or limited information is available about other Pleurotus species. The purpose of our study was to compare the decolorization capacity of several Pleurotus species with the aim to find new efficient synthetic dye-decolorizing strains. On the basis of the results obtained from a simple screening test with two different dyes (one azo and one anthraquinone), we focused our attention on P. calyptratus, which has not attracted the interest of researchers, although it possesses good decolorization abilities and could be promising for further biotechnological applications.
2. Material and methods 2.1. Organisms All studied species (see Table 1) were obtained from the CCBAS collection (Institute of Microbiology AS CR, Prague, Czech Republic). The cultures were maintained by serial transfers and kept on wort agar slants at 4 8C.
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Table 1 Decolorization and growth characteristics of the set of Pleurotus species after 10 days of cultivation on Petri dishes Species
Orange G Growth
Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus
calyptratus CCBAS 461 citrinopileatus CCBAS 691 cornucopiae CCBAS 464 cystidiosus CCBAS 466 dryinus CCBAS 468 eryngii CCBAS 471 ostreatus CCBAS 473 pulmonarius CCBAS 479
a
75.6 61.1 33.3 27.8 11.1 41.1 75.5 94.4
RBBR Decolorization 55.0 0 33.3 0 0 0 51.1 94.4
b
(10/14) (10/22)
(3/14) (3/12)
Growth a
Decolorization b
38.9 20.0 26.9 37.8 14.4 35.6 94.4 55.5
48.9 0 30.6 27.8 16.7 40.0 86.6 55.5
(3/28) (10/30) (3/30) (6/40) (3/30) (3/14) (3/18)
a
Growth: the number represents the diameter of the mycelial colony (measured on the 10th day of cultivation) in % of the whole Petri dish diameter (90 mm). Decolorization: the first number represents the diameter of the decolorized zone (measured on the 10th day of cultivation) in % of the whole Petri dish diameter (90 mm); the number in parentheses indicates the day of cultivation on which the decolorization started/the day of cultivation on which the Petri dish was completely decolorized. b
2.2. Chemicals 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-methyl2-benzothiazolinone hydrazone (MBTH), 3-dimethylaminobenzoic acid (DMAB), Remazol Brilliant Blue R (RBBR), Crystal Violet and Malachite Green were purchased from Sigma, Orange G, Amaranth, Poly R-478 and Cuphthalocyanin from Fluka and veratryl alcohol (3,4-dimethoxybenzyl alcohol) from Aldrich. All chemicals were of analytical grade.
2.3. Culture conditions Static cultivation was carried out at 25 8C in 100 ml Erlenmeyer flasks with 20 ml of N-limited (0.2 g/1 of ammonium tartrate) Kirk medium [14] or of Malt extract medium (malt extract 1% and glucose 1%) or with these media supplemented with the respective dyes at a final concentration of 500 ppm (Orange G, RBBR, Amaranth and Cu-phthalocyanin) or 50 ppm (Malachite Green and Crystal Violet). The flasks were inoculated with two wort agar plugs (10 mm diameter) cut from an actively growing part of a colony on a Petri dish, and incubated at 27 8C for 20 days. Decolorization and enzyme production were measured on the 3rd, 5th, 7th, 10th, 14th, 18th and 20th day of cultivation. Cultivation on solid media was carried out on Petri dishes (90 mm diameter) containing N-limited Kirk medium with Orange G or RBBR at a final concentration of 200 ppm. Plates (four parallels) were inoculated with mycelial plugs (3 mm diameter) cut from actively growing mycelia.
2.4. Decolorization assays Decolorization of the liquid medium was measured in the filtrates (four parallel flasks) after removing the mycelia and monitored spectrophotometrically at the maximum visible wavelength of absorbance (478 nm for Orange G, 595 nm for RBBR, 525 nm for Amaranth, 625 nm for Cu-phthalocyanin, 521 nm for Poly R-478, 616 nm for Malachite Green and 591 nm for Crystal Violet). Systems without the fungus served as an abiotic control. The dye sorption effect of mycelia during the decolorization process was determined using the biotic control according to [15]. Decolorization activity was also tested on solid media, where the radial growth (measured as the diameter of the colonies) and the zone of color change (measured as the diameter of the decolorized zone) on the agar plates were measured daily. All measurements were repeated three times.
2.5. Ligninolytic enzyme assays Enzyme activity was measured in filtrates from four parallel flasks detained after mycelia removal. Activities of extracellular laccase (EC 1.10.3.2, Lac) and manganese peroxidase (EC 1.11.1.13, MnP) were determined spectrophotometrically by monitoring the absorbance increase at 425 nm (laccase) or 590 nm
(MnP) in the reaction mixture. Lac activity was assayed according to [16] by monitoring the oxidation of ABTS. Determination of MnP activity using MBTH and DMAB was based on the method of [17] modified according to [18]. MBTH and DMAB were oxidatively coupled by the action of the enzyme in the presence of added H2O2 and Mn2+ ions to give a purple indamine dye product. The values were corrected for the activities in the test samples without manganese (non-specific peroxidase activity), where manganese sulfate was substituted by ethylenediaminetetraacetate (EDTA) to chelate Mn2+ ions present in the extract. Aryl alcohol oxidase (EC 1.1.3.7, AAO) activity was measured spectrophotometrically as the oxidation of veratryl alcohol to veratraldehyde at 310 nm according to [19]. Activity of lignin peroxidase (LiP) was determined spectrophotometrically at 310 nm by monitoring the oxidation of veratryl alcohol in the presence of H2O2 according to [20]. All measurements were repeated three times. One unit of enzyme activity (U) was defined as an amount of enzyme catalyzing the production of one micromole of reaction product per ml per min.
3. Results and discussion 3.1. Decolorization ability and ligninolytic enzyme production of different Pleurotus species A simple agar-plate test with two structurally different dyes (Orange G, azo dye; RBBR, anthraquinone dye) was used for determining the decolorization capability of eight different Pleurotus species (Table 1). Four species were able to decolorize both tested dyes, three species decolorized only RBBR and one strain (Pleurotus citrinopileatus CCBAS 691) did not show any decolorization ability. None of the species decolorized only Orange G. The results indicate that azo dyes are more resistant to decolorization than anthraquinone dyes. These findings are in correspondence with the literature data [2,6]. Among the studied species, P. ostreatus and P. pulmonarius exhibited the highest decolorization capacity. The decolorization process in both species started very early (on the 3rd day of cultivation) and after 12–18 days the dye in the Petri dish was decolorized completely. Moreover, in P. ostreatus and P. pulmonarius we found the highest growth rate on media in the presence of the tested dyes. Nevertheless, these two species have already been studied in detail by many other researchers and their ability to efficiently decolorize synthetic dyes is well known [8–11]. Our results revealed that P.
I. Eichlerova´ et al. / Process Biochemistry 41 (2006) 941–946
calyptratus CCBAS 461 was another Pleurotus strain, which was able to grow quite well on the medium with synthetic dyes and to decolorize both Orange G and RBBR (see Table 1). In this strain the decolorization was about 50% within 10 days and after 14–28 days the decolorization was complete. Surprisingly, only very little attention has been paid to this species in the literature and we did not find any information on its decolorization ability. Considering that decolorization abilities of white rot fungi are assumed to be connected with their ligninolytic properties [21], we tested Lac, MnP, AAO, LiP and overall hydrogen peroxide production in the set of Pleurotus species (Table 2). Our data did not show any significant correlation between the tested characteristics and the decolorization capacity, but the set of species was too small to permit any conclusion about the role of individual enzymes in decolorization processes. All the tested species produced Lac and MnP, but no detectable amount of LiP. Five species (P. calyptratus, P. citrinopileatus, P. eryngii, P. ostreatus and P. pulmonarius) produced aryl alcohol oxidase. The production of this enzyme is well known in P. eryngii [22,23] and also there are some literature reports on AAO production in P. pulmonarius [24] and in P. ostreatus [25]. Varela et al. [26] confirmed the presence of gene aao encoding aryl alcohol oxidase in P. eryngii, P. ostreatus and P. pulmonarius; nevertheless, the composition of the culture medium substantially affect the expression of this enzyme. Interestingly, we did not found any literature data on AAO production in P. calyptratus and P. citrinopileatus. In the case of P. citrinopileatus, which did not decolorize any dye tested, we detected relatively high production of AAO, which was comparable with P. eryngii, but the lowest Lac production among the studied Pleurotus species. We found lower Lac activity also in some other Pleurotus species, which were able to decolorize only RBBR but not Orange G. This finding indicates a certain role of Lac in the decolorization process, which has been already described by many authors [27,28]. On the other hand, the species with the highest Lac and AAO production (P. eryngii) did not show the best decolorization properties and was also able to decolorize only RBBR. All the species decolorizing only RBBR showed good production of MnP. These findings are in correspondence with the literature data, which emphasize the importance of MnP in decolorization of RBBR or other anthraquinone dyes [29,30]. Surprisingly, Pleurotus species able to decolorize both tested dyes produced
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mostly low amounts of MnP, which was probably compensated by their higher production of Lac. Moreover, some of these species produced AAO. This suggests the synergistic action of different enzyme types in the decolorization process. It is well known that also different mediators, radicals, hydrogen peroxide etc. take part in decolorization processes [31–33], but our studies did not reveal any correlation between the overall H2O2 production and the decolorization properties of Pleurotus species. We found a very high production of H2O2 in the species with no (P. citrinopileatus) or very low (P. dryinus) decolorization ability and also in the species with a high decolorization capacity (P. ostreatus). In all other species tested the H2O2 production was almost the same—approximately three times lower than in P. citrinopileatus or P. dryinus. 3.2. Decolorization of synthetic dyes by P. calyptratus in liquid media On the basis of the above results (see Tables 1 and 2) we focused our attention on P. calyptratus, strain CCBAS 461, which in spite of its quite good decolorization abilities and sufficient production of Lac, MnP and even AAO, has not been studied by other researchers. In liquid culture, P. calyptratus was able to decolorize several synthetic dyes belonging to different chemical groups. In the present work we tested its ability to decolorize Orange G and Amaranth (azo dyes), RBBR (anthraquinone dye), Cuphthalocyanin (phthalocyanine dye), Poly R-478 (polyaromatic dye), Crystal Violet and Malachite Green (triphenylmethane dyes) during 14 days of cultivation in Kirk medium. As expected, the dyes belonging to chemically different groups were not decolorized to the same extent (Fig. 1). Malachite Green, Crystal Violet and Poly R-478 were decolorized only poorly (after 14 days the extents of decolorization were only 5, 27 and 10% of the dye, respectively), while Amaranth was decolorized quite efficiently (50% of the dye was removed after 14 days); Orange G, RBBR and Cu-phthalocyanin were decolorized to a very high extent (91, 78 and 75% respectively). Nyanhongo et al. [28] and Xu [34] also refer to low triphenylmethane dye decolorization capacity in white rot fungi. A good ability to decolorize anthraquinone dyes (RBBR) and phthalocyanine dyes (Cu-phthalocyanin) by white rot fungi was mentioned in several papers (e.g. [2,7]). The easy decolorization of the azo dyes Orange G and Amaranth is
Table 2 Ligninolytic enzyme and overall hydrogen peroxide production of Pleurotus species cultivated in Kirk medium Species
Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus
Maximum of production
calyptratus CCBAS 461 citrinopileatus CCBAS 691 cornucopiae CCBAS 464 cystidiosus CCBAS 466 dryinus CCBAS 468 eryngii CCBAS 471 ostreatus CCBAS 473 pulmonarius CCBAS 479
Laccase (U/L)
MnP (U/L)
AAO (U/L)
H2O2 (mM)
31.1 2.8 5.5 0.06 73.1 8.4 13.3 1.0 14.0 1.2 108.6 12.0 73.0 6.5 47.5 4.5
1.32 0.2 0.68 0.08 0.11 0.01 0.92 0.07 1.40 0.11 1.02 0.1 0.69 0.06 0.10 0.02
23.82 3.5 46.37 5.5 0 0 0 124.14 14.1 24.80 2.2 18.03 1.5
0.48 0.05 1.73 0.20 0.43 0.02 0.46 0.03 1.35 0.11 0.46 0.06 1.58 0.17 0.36 0.04
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3.3. Production of Lac and MnP by P. calyptratus cultivated in media with synthetic dyes
Fig. 1. Decolorization of different dyes by P. calyptratus cultivated in liquid Kirk medium.
somewhat surprising, because azo dyes are, due to their chemical structure, considered to be resistant to decolorization. Our findings imply that although structural differences in the molecule of the dye can affect the decolorization process, the overall complexity of structure alone is not necessarily an indicator of an unsuccessful decolorization. Out of the studied dyes, Orange G and RBBR were decolorized most efficiently and therefore they were chosen for our further work. Moreover, azo and anthraquinone dyes represent the main chemical dye groups. P. calyptratus efficiently decolorized Orange G and RBBR in two different liquid media—Kirk medium and Malt extract medium (Fig. 2). Our results showed that while in Kirk medium the decolorization of Orange G was very efficient (91% of the dye was removed within 14 days), the decolorization in Malt extract medium was lower (42%). On the other hand, RBBR was decolorized to a slightly higher extent in Malt extract medium (85% of the dye was removed in 14 days) than in Kirk medium (78%). It is interesting that RBBR was decolorized more rapidly than Orange G in both media at the very beginning of the cultivation, while later on the decolorization rate became almost the same for both dyes. These findings support our conviction that the composition of the media substantially influences the decolorization process in fungi; this opinion is shared by other authors [35–37].
Production of Lac and MnP—the main ligninolytic enzymes in P. calyptratus—was significantly influenced by the composition of cultivation media and by the presence of the dye (Fig. 3). P. calyptratus produces a substantially higher amount (more than four times in a maximum) of Lac in Malt extract medium than in Kirk medium. Also MnP production was higher in Malt extract medium than in Kirk medium. These differences were probably caused by different nitrogen sources in the media tested. In connection with the decolorization processes several authors [35–37] emphasized the influence of different nutrition sources, especially the concentration and type of nitrogen sources, on ligninolytic enzyme production. The presence of the dyes negatively influenced the production of ligninolytic enzymes almost in all cases. The exception was Lac activity in Malt extract medium, which increased when Orange G was present. This is somewhat surprising because a more efficient Orange G decolorization was found in Kirk medium. Similar results were obtained when we checked the specific activity of the studied enzymes. Nevertheless, one has to take into account the fact that decolorization of synthetic dyes is more complicated and ligninolytic enzymes are not the only factors that take part in this process. In conclusion, our results showed that P. calyptratus is able to decolorize efficiently several synthetic dyes belonging to different chemical groups. The highest decolorization capacity was found with Orange G and RBBR. On comparing the two media tested, we detected a more rapid Orange G decolorization in Kirk medium, while RBBR was decolorized to a higher extent in Malt extract medium. The strain produced a relatively high amount of Lac, MnP and also aryl-alcohol oxidase, which was not in P. calyptratus mentioned by other authors up to now. Production of Lac and MnP was higher in Malt extract medium, but it was reduced by the presence of the dyes in both media. Our findings could contribute to a better knowledge of the properties of P. calyptratus, which has as yet not been studied in detail and its decolorization abilities could be promising for further biotechnological applications.
Fig. 2. Decolorization of Orange G and RBBR by P. calyptratus cultivated in liquid media.
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Fig. 3. Lac and MnP production of P. calyptratus during cultivation in liquid media.
Acknowledgements This work was supported by grant no. 206/02/D119 from the Grant Agency of the Czech Republic and by Institutional Research Concept no. AV0Z50200510.
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Short communication
Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461 Ivana Eichlerova´ *, Ladislav Homolka, Frantisˇek Nerud Institute of Microbiology AS CR, Vı´deo`ska´ 1083, 142 20 Prague 4, Czech Republic Received 9 June 2005; received in revised form 14 September 2005; accepted 13 October 2005
Abstract Eight different Pleurotus species were tested for their Orange G and Remazol Brilliant Blue R (RBBR) decolorization capacity and their ligninolytic properties. All the species produced laccase (Lac) and manganese peroxidase (MnP) and in five species we found aryl-alcohol oxidase (AAO) activity. Strain CCBAS 461 of a very little studied species Pleurotus calyptratus was chosen for a more detailed study. This strain produced a relatively high amount of Lac, MnP and also AAO. Within 14 days the strain decolorized up to 91% of Orange G and 85% of RBBR in liquid culture and more than 50% of these dyes on agar plates. P. calyptratus is able to decolorize efficiently also other azo and phthalocyanine dyes, but only a limited decolorization capacity was found in the case of polyaromatic and triphenylmethane dyes. Lac and MnP production was strongly influenced by the kind of cultivation media and by the dye present. # 2005 Elsevier Ltd. All rights reserved. Keywords: Decolorization; Synthetic dyes; Pleurotus calyptratus; Laccase; Manganese peroxidase; Aryl-alcohol oxidase
1. Introduction Dye wastewater from textile and dyestuff industries is very difficult to treat. Synthetic dyes, classified by their chromophore as azo, anthraquinone, triphenylmethane, heterocyclic or phthalocyanine dyes, are very stable and resistant to microbial attack and therefore it is difficult to remove them from effluents by conventional biological processes. Decolorization of industrial dyes can be achieved by physico-chemical methods, such as adsorption, precipitation or chemical degradation, but these are very expensive, which limits their application [1]. The decolorization of the dyes through intermediates of different color has been proposed to be a series of multiple reactions. In case of anaerobic decolorization of azo dyes by some bacteria, arising intermediates and final products are carcinogenic and mostly more toxic than the starting dyes. In recent years, the utilization of biodegradative abilities of some white rot fungi seems to be promising [2]. Owing to their extracellular nonspecific free radical-based enzymatic system they can completely eliminate a variety of xenobiotics, including synthetic dyes, giving rise to non-toxic compounds [3,4].
* Corresponding author. Tel.: +42 241062611; fax: +42 241062384. E-mail address: [email protected] (I. Eichlerova´). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.018
Many white rot fungi (e.g. Phanerochaete chrysosporium, Bjerkandera adusta, Trametes versicolor etc.) have been intensively studied in connection with the decolorization processes [5–7]. Many papers described also biodegradative and decolorization abilities of several species of the genus Pleurotus, especially P. ostreatus [8,9], P. pulmonarius [10,11], P. eryngii [12] or P. sajor-caju [13]. Nevertheless, no or limited information is available about other Pleurotus species. The purpose of our study was to compare the decolorization capacity of several Pleurotus species with the aim to find new efficient synthetic dye-decolorizing strains. On the basis of the results obtained from a simple screening test with two different dyes (one azo and one anthraquinone), we focused our attention on P. calyptratus, which has not attracted the interest of researchers, although it possesses good decolorization abilities and could be promising for further biotechnological applications.
2. Material and methods 2.1. Organisms All studied species (see Table 1) were obtained from the CCBAS collection (Institute of Microbiology AS CR, Prague, Czech Republic). The cultures were maintained by serial transfers and kept on wort agar slants at 4 8C.
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Table 1 Decolorization and growth characteristics of the set of Pleurotus species after 10 days of cultivation on Petri dishes Species
Orange G Growth
Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus
calyptratus CCBAS 461 citrinopileatus CCBAS 691 cornucopiae CCBAS 464 cystidiosus CCBAS 466 dryinus CCBAS 468 eryngii CCBAS 471 ostreatus CCBAS 473 pulmonarius CCBAS 479
a
75.6 61.1 33.3 27.8 11.1 41.1 75.5 94.4
RBBR Decolorization 55.0 0 33.3 0 0 0 51.1 94.4
b
(10/14) (10/22)
(3/14) (3/12)
Growth a
Decolorization b
38.9 20.0 26.9 37.8 14.4 35.6 94.4 55.5
48.9 0 30.6 27.8 16.7 40.0 86.6 55.5
(3/28) (10/30) (3/30) (6/40) (3/30) (3/14) (3/18)
a
Growth: the number represents the diameter of the mycelial colony (measured on the 10th day of cultivation) in % of the whole Petri dish diameter (90 mm). Decolorization: the first number represents the diameter of the decolorized zone (measured on the 10th day of cultivation) in % of the whole Petri dish diameter (90 mm); the number in parentheses indicates the day of cultivation on which the decolorization started/the day of cultivation on which the Petri dish was completely decolorized. b
2.2. Chemicals 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-methyl2-benzothiazolinone hydrazone (MBTH), 3-dimethylaminobenzoic acid (DMAB), Remazol Brilliant Blue R (RBBR), Crystal Violet and Malachite Green were purchased from Sigma, Orange G, Amaranth, Poly R-478 and Cuphthalocyanin from Fluka and veratryl alcohol (3,4-dimethoxybenzyl alcohol) from Aldrich. All chemicals were of analytical grade.
2.3. Culture conditions Static cultivation was carried out at 25 8C in 100 ml Erlenmeyer flasks with 20 ml of N-limited (0.2 g/1 of ammonium tartrate) Kirk medium [14] or of Malt extract medium (malt extract 1% and glucose 1%) or with these media supplemented with the respective dyes at a final concentration of 500 ppm (Orange G, RBBR, Amaranth and Cu-phthalocyanin) or 50 ppm (Malachite Green and Crystal Violet). The flasks were inoculated with two wort agar plugs (10 mm diameter) cut from an actively growing part of a colony on a Petri dish, and incubated at 27 8C for 20 days. Decolorization and enzyme production were measured on the 3rd, 5th, 7th, 10th, 14th, 18th and 20th day of cultivation. Cultivation on solid media was carried out on Petri dishes (90 mm diameter) containing N-limited Kirk medium with Orange G or RBBR at a final concentration of 200 ppm. Plates (four parallels) were inoculated with mycelial plugs (3 mm diameter) cut from actively growing mycelia.
2.4. Decolorization assays Decolorization of the liquid medium was measured in the filtrates (four parallel flasks) after removing the mycelia and monitored spectrophotometrically at the maximum visible wavelength of absorbance (478 nm for Orange G, 595 nm for RBBR, 525 nm for Amaranth, 625 nm for Cu-phthalocyanin, 521 nm for Poly R-478, 616 nm for Malachite Green and 591 nm for Crystal Violet). Systems without the fungus served as an abiotic control. The dye sorption effect of mycelia during the decolorization process was determined using the biotic control according to [15]. Decolorization activity was also tested on solid media, where the radial growth (measured as the diameter of the colonies) and the zone of color change (measured as the diameter of the decolorized zone) on the agar plates were measured daily. All measurements were repeated three times.
2.5. Ligninolytic enzyme assays Enzyme activity was measured in filtrates from four parallel flasks detained after mycelia removal. Activities of extracellular laccase (EC 1.10.3.2, Lac) and manganese peroxidase (EC 1.11.1.13, MnP) were determined spectrophotometrically by monitoring the absorbance increase at 425 nm (laccase) or 590 nm
(MnP) in the reaction mixture. Lac activity was assayed according to [16] by monitoring the oxidation of ABTS. Determination of MnP activity using MBTH and DMAB was based on the method of [17] modified according to [18]. MBTH and DMAB were oxidatively coupled by the action of the enzyme in the presence of added H2O2 and Mn2+ ions to give a purple indamine dye product. The values were corrected for the activities in the test samples without manganese (non-specific peroxidase activity), where manganese sulfate was substituted by ethylenediaminetetraacetate (EDTA) to chelate Mn2+ ions present in the extract. Aryl alcohol oxidase (EC 1.1.3.7, AAO) activity was measured spectrophotometrically as the oxidation of veratryl alcohol to veratraldehyde at 310 nm according to [19]. Activity of lignin peroxidase (LiP) was determined spectrophotometrically at 310 nm by monitoring the oxidation of veratryl alcohol in the presence of H2O2 according to [20]. All measurements were repeated three times. One unit of enzyme activity (U) was defined as an amount of enzyme catalyzing the production of one micromole of reaction product per ml per min.
3. Results and discussion 3.1. Decolorization ability and ligninolytic enzyme production of different Pleurotus species A simple agar-plate test with two structurally different dyes (Orange G, azo dye; RBBR, anthraquinone dye) was used for determining the decolorization capability of eight different Pleurotus species (Table 1). Four species were able to decolorize both tested dyes, three species decolorized only RBBR and one strain (Pleurotus citrinopileatus CCBAS 691) did not show any decolorization ability. None of the species decolorized only Orange G. The results indicate that azo dyes are more resistant to decolorization than anthraquinone dyes. These findings are in correspondence with the literature data [2,6]. Among the studied species, P. ostreatus and P. pulmonarius exhibited the highest decolorization capacity. The decolorization process in both species started very early (on the 3rd day of cultivation) and after 12–18 days the dye in the Petri dish was decolorized completely. Moreover, in P. ostreatus and P. pulmonarius we found the highest growth rate on media in the presence of the tested dyes. Nevertheless, these two species have already been studied in detail by many other researchers and their ability to efficiently decolorize synthetic dyes is well known [8–11]. Our results revealed that P.
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calyptratus CCBAS 461 was another Pleurotus strain, which was able to grow quite well on the medium with synthetic dyes and to decolorize both Orange G and RBBR (see Table 1). In this strain the decolorization was about 50% within 10 days and after 14–28 days the decolorization was complete. Surprisingly, only very little attention has been paid to this species in the literature and we did not find any information on its decolorization ability. Considering that decolorization abilities of white rot fungi are assumed to be connected with their ligninolytic properties [21], we tested Lac, MnP, AAO, LiP and overall hydrogen peroxide production in the set of Pleurotus species (Table 2). Our data did not show any significant correlation between the tested characteristics and the decolorization capacity, but the set of species was too small to permit any conclusion about the role of individual enzymes in decolorization processes. All the tested species produced Lac and MnP, but no detectable amount of LiP. Five species (P. calyptratus, P. citrinopileatus, P. eryngii, P. ostreatus and P. pulmonarius) produced aryl alcohol oxidase. The production of this enzyme is well known in P. eryngii [22,23] and also there are some literature reports on AAO production in P. pulmonarius [24] and in P. ostreatus [25]. Varela et al. [26] confirmed the presence of gene aao encoding aryl alcohol oxidase in P. eryngii, P. ostreatus and P. pulmonarius; nevertheless, the composition of the culture medium substantially affect the expression of this enzyme. Interestingly, we did not found any literature data on AAO production in P. calyptratus and P. citrinopileatus. In the case of P. citrinopileatus, which did not decolorize any dye tested, we detected relatively high production of AAO, which was comparable with P. eryngii, but the lowest Lac production among the studied Pleurotus species. We found lower Lac activity also in some other Pleurotus species, which were able to decolorize only RBBR but not Orange G. This finding indicates a certain role of Lac in the decolorization process, which has been already described by many authors [27,28]. On the other hand, the species with the highest Lac and AAO production (P. eryngii) did not show the best decolorization properties and was also able to decolorize only RBBR. All the species decolorizing only RBBR showed good production of MnP. These findings are in correspondence with the literature data, which emphasize the importance of MnP in decolorization of RBBR or other anthraquinone dyes [29,30]. Surprisingly, Pleurotus species able to decolorize both tested dyes produced
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mostly low amounts of MnP, which was probably compensated by their higher production of Lac. Moreover, some of these species produced AAO. This suggests the synergistic action of different enzyme types in the decolorization process. It is well known that also different mediators, radicals, hydrogen peroxide etc. take part in decolorization processes [31–33], but our studies did not reveal any correlation between the overall H2O2 production and the decolorization properties of Pleurotus species. We found a very high production of H2O2 in the species with no (P. citrinopileatus) or very low (P. dryinus) decolorization ability and also in the species with a high decolorization capacity (P. ostreatus). In all other species tested the H2O2 production was almost the same—approximately three times lower than in P. citrinopileatus or P. dryinus. 3.2. Decolorization of synthetic dyes by P. calyptratus in liquid media On the basis of the above results (see Tables 1 and 2) we focused our attention on P. calyptratus, strain CCBAS 461, which in spite of its quite good decolorization abilities and sufficient production of Lac, MnP and even AAO, has not been studied by other researchers. In liquid culture, P. calyptratus was able to decolorize several synthetic dyes belonging to different chemical groups. In the present work we tested its ability to decolorize Orange G and Amaranth (azo dyes), RBBR (anthraquinone dye), Cuphthalocyanin (phthalocyanine dye), Poly R-478 (polyaromatic dye), Crystal Violet and Malachite Green (triphenylmethane dyes) during 14 days of cultivation in Kirk medium. As expected, the dyes belonging to chemically different groups were not decolorized to the same extent (Fig. 1). Malachite Green, Crystal Violet and Poly R-478 were decolorized only poorly (after 14 days the extents of decolorization were only 5, 27 and 10% of the dye, respectively), while Amaranth was decolorized quite efficiently (50% of the dye was removed after 14 days); Orange G, RBBR and Cu-phthalocyanin were decolorized to a very high extent (91, 78 and 75% respectively). Nyanhongo et al. [28] and Xu [34] also refer to low triphenylmethane dye decolorization capacity in white rot fungi. A good ability to decolorize anthraquinone dyes (RBBR) and phthalocyanine dyes (Cu-phthalocyanin) by white rot fungi was mentioned in several papers (e.g. [2,7]). The easy decolorization of the azo dyes Orange G and Amaranth is
Table 2 Ligninolytic enzyme and overall hydrogen peroxide production of Pleurotus species cultivated in Kirk medium Species
Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus Pleurotus
Maximum of production
calyptratus CCBAS 461 citrinopileatus CCBAS 691 cornucopiae CCBAS 464 cystidiosus CCBAS 466 dryinus CCBAS 468 eryngii CCBAS 471 ostreatus CCBAS 473 pulmonarius CCBAS 479
Laccase (U/L)
MnP (U/L)
AAO (U/L)
H2O2 (mM)
31.1 2.8 5.5 0.06 73.1 8.4 13.3 1.0 14.0 1.2 108.6 12.0 73.0 6.5 47.5 4.5
1.32 0.2 0.68 0.08 0.11 0.01 0.92 0.07 1.40 0.11 1.02 0.1 0.69 0.06 0.10 0.02
23.82 3.5 46.37 5.5 0 0 0 124.14 14.1 24.80 2.2 18.03 1.5
0.48 0.05 1.73 0.20 0.43 0.02 0.46 0.03 1.35 0.11 0.46 0.06 1.58 0.17 0.36 0.04
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3.3. Production of Lac and MnP by P. calyptratus cultivated in media with synthetic dyes
Fig. 1. Decolorization of different dyes by P. calyptratus cultivated in liquid Kirk medium.
somewhat surprising, because azo dyes are, due to their chemical structure, considered to be resistant to decolorization. Our findings imply that although structural differences in the molecule of the dye can affect the decolorization process, the overall complexity of structure alone is not necessarily an indicator of an unsuccessful decolorization. Out of the studied dyes, Orange G and RBBR were decolorized most efficiently and therefore they were chosen for our further work. Moreover, azo and anthraquinone dyes represent the main chemical dye groups. P. calyptratus efficiently decolorized Orange G and RBBR in two different liquid media—Kirk medium and Malt extract medium (Fig. 2). Our results showed that while in Kirk medium the decolorization of Orange G was very efficient (91% of the dye was removed within 14 days), the decolorization in Malt extract medium was lower (42%). On the other hand, RBBR was decolorized to a slightly higher extent in Malt extract medium (85% of the dye was removed in 14 days) than in Kirk medium (78%). It is interesting that RBBR was decolorized more rapidly than Orange G in both media at the very beginning of the cultivation, while later on the decolorization rate became almost the same for both dyes. These findings support our conviction that the composition of the media substantially influences the decolorization process in fungi; this opinion is shared by other authors [35–37].
Production of Lac and MnP—the main ligninolytic enzymes in P. calyptratus—was significantly influenced by the composition of cultivation media and by the presence of the dye (Fig. 3). P. calyptratus produces a substantially higher amount (more than four times in a maximum) of Lac in Malt extract medium than in Kirk medium. Also MnP production was higher in Malt extract medium than in Kirk medium. These differences were probably caused by different nitrogen sources in the media tested. In connection with the decolorization processes several authors [35–37] emphasized the influence of different nutrition sources, especially the concentration and type of nitrogen sources, on ligninolytic enzyme production. The presence of the dyes negatively influenced the production of ligninolytic enzymes almost in all cases. The exception was Lac activity in Malt extract medium, which increased when Orange G was present. This is somewhat surprising because a more efficient Orange G decolorization was found in Kirk medium. Similar results were obtained when we checked the specific activity of the studied enzymes. Nevertheless, one has to take into account the fact that decolorization of synthetic dyes is more complicated and ligninolytic enzymes are not the only factors that take part in this process. In conclusion, our results showed that P. calyptratus is able to decolorize efficiently several synthetic dyes belonging to different chemical groups. The highest decolorization capacity was found with Orange G and RBBR. On comparing the two media tested, we detected a more rapid Orange G decolorization in Kirk medium, while RBBR was decolorized to a higher extent in Malt extract medium. The strain produced a relatively high amount of Lac, MnP and also aryl-alcohol oxidase, which was not in P. calyptratus mentioned by other authors up to now. Production of Lac and MnP was higher in Malt extract medium, but it was reduced by the presence of the dyes in both media. Our findings could contribute to a better knowledge of the properties of P. calyptratus, which has as yet not been studied in detail and its decolorization abilities could be promising for further biotechnological applications.
Fig. 2. Decolorization of Orange G and RBBR by P. calyptratus cultivated in liquid media.
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Fig. 3. Lac and MnP production of P. calyptratus during cultivation in liquid media.
Acknowledgements This work was supported by grant no. 206/02/D119 from the Grant Agency of the Czech Republic and by Institutional Research Concept no. AV0Z50200510.
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