AGAR-PLATE SCREENING FOR TEXTILE DYE DECOLORISATION BY WHITE ROT FUNGI Pleurotus SPECIES (Pleurotus cornucopiae var.

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1 by PSP Volume 16 No Fresenius Environmental Bulletin AGAR-PLATE SCREENING FOR TEXTILE DYE DECOLORISATION BY WHITE ROT FUNGI Pleurotus SPECIES (Pleurotus cornucopiae var.citrinopileatus, P. djamor, P. eryngii, P. ostreatus and P. sajor-caju). Erbil Kalmış 1*, Nuri Azbar 1 and Fatih Kalyoncu 2 1 Ege University, Faculty of Engineering, Bioengineering Department, 35 Bornova, Izmir, Turkey 2 Celal Bayar University, Faculty of Science & Arts, Department of Biology, 451 Muradiye, Manisa,Turkey SUMMARY A screening test for five different white rot fungal strains (Pleurotus species: Pleurotus cornucopiae var. citrinopileatus, P. djamor, P. eryngii, P. ostreatus and P. sajorcaju) was carried out to assess their decolorisation capacities for five different textile dyestuffs, namely Indanthren yellow F3GC Collosiol (IYFC), Blue CC Dranix (BCCD), Indanthren Blue CLF Collosiol (IB), Remazol Brilliant Blue BB (RB), and Levafix Brilliant Blue E-B (LBB) on agar plates. Full decolorisation was observed only for RB and LBB (dye concentrations <2 mg L -1 ). Similar to LBB dye, IB was also not decolorized at all dye concentrations above 2 mg L -1, but the original color of the dye was converted into yellow below 2 mg L -1 by all organisms used. None of the organism used in this study was able to fully decolorize IYFC dye, but conversion of original color of the dye into brown was observed for all dye concentrations. In addition, no decolorisation was observed for BCCD at all dye concentrations used. In terms of radial growth, in most cases, low dye concentrations (< 2 mg L -1 ) were well tolerated by the organisms used, except for some of them, showing either retardation or full inhibition in growth. KEYWORDS: white rot fungi, biological decolorisation, textile dye, agar plate screening. INTRODUCTION Recently, new and tighter regulations coupled with increased enforcement concerning wastewater discharges have been forced in many countries. This tight legislation, in conjunction with international trade pressures, such as increasing competition and the introduction of eco-labels for textile products on the European and US markets, has been threatening the very survival of the textile industry in many industrialized countries. Commonly applied treatment methods for color removal from dye-contaminated effluents consist of integrated processes involving various combinations of biological, physical and chemical decolorisation methods [1-3]. These integrated treatment methods are efficient but not cost-effective. Conventional wastewater treatment plants (WWTP) relying on activated sludge systems are not adequate for the treatment of textile mill effluents, neither on site nor after dilution with domestic wastewater at the sewage works. Activated sludge and other types of bioreactors fail to remove sufficiently color, COD, N, surfactants and other micro-pollutants present in the textile effluents [4, 5]. Tertiary coagulation/flocculation is often used with variable results, but nowadays nearly complete decolorisation and water reuse are possible, whereas sludge disposal remains, however, a problem. Ozone is increasingly used as a final step, but costs and aldehyde formation prevent its wider acceptance. Membrane filtration of process substreams may yield substantial cost savings by allowing reuse of water, chemicals and heat. Handling and disposal of the concentrated stream remains, however, a severe limitation to filtration processes. In view of the need for a technically and economically satisfying treatment technology, a flurry of emerging technologies (e.g. biologically activated GAC filtration, foam floatation, electrolysis, photocatalysis, biosorption and Fenton oxidation) are being proposed and tested at different stages of commercialization. Many researches related on determination of decolorisation capacity of fungi have been reported from different work groups around the world [6-9]. This study, in which 5 different strains of white rot fungi were examined for the decolorisation of five textile dyes (Indanthren yellow F3GC Collosiol (IYFC), Blue CC Dranix (BCCD), Indanthren Blue CLF Collosiol (C.I. Vat Blue 66) (IB), Remazol Brilliant Blue (C.I. Reactive Blue 19) (RB), Levafix Brilliant Blue EB (C.I. Reactive Blue 29) (LBB)), is part of 139

2 by PSP Volume 16 No Fresenius Environmental Bulletin a research project which was focused on determination of decolorisation potential of fungi isolated from contaminated soil. The purpose of this study was to find other efficient fungal degraders using a screening method, and characterize their dye degradation capacity comparatively for various dyes. For this purpose, we focused our effort on the strains, which have attracted only little or no research attention, with those which were already studied extensively for comparison purpose. MATERIALS AND METHODS Fungal strains The screening test was carried out with 5 strains of white rot fungi, namely, Pleurotus cornucopiae var. citrinopileatus, P. dijamor, P. eryngii, P. ostreatus and P. sajor-caju. All these strains were obtained from a local spawn producer, Agromycel company Denizli, Turkey ( They were deposited in the Culture Collection of the Department of Bioengineering, Faculty of Engineering, Ege University, Turkey, and maintained on malt extract agar slants at 4 o C until use. Culture conditions and Experimental set-up All the strains were inoculated on agar plates (9 mm in diameter, 2 ml medium/petri-dish) containing modified Kirk s basal salts media with the following composition: glucose 1 g,.36 g urea, 2 g KH 2 PO 4,.5 g MgSO 4.7H 2 O,.99 g CaCl 2 in ml distilled water. Agar plates were supplemented with one of five different dyes corresponding to dye concentrations of 2, 3,, 5,,,, 2, mg L -1 for each dye (only the results for dye concentrations of 2, 5, 2, mg L -1 were reported here), except for LBB, which was used at concentrations of 5, 1 and 2 mg L -1 due to its toxicity at higher concentrations on fungal growth. Inoculums consisted of 6-mm agar plugs of one week-old cultures grown on modified Kirk s basal medium at 26 o C. The plug cut under sterile conditions from the outer edge of the agar plate was transferred onto the centre of the experimental plates for each replicate. A control plate with no dye added was also inoculated with each strain. In addition, not inoculated plates served as controls for abiotic decolorisation. Each fungus was tested in three independent experiments on all plates. The plates were incubated at 26± 1 o C for at least 3 days. Mycelial growth was followed by measuring radial extension of the mycelium, as described by [1], with a caliper gauge along two diameters at right angles to one another and the average diameter for each plate calculated. The mean mycelial growth was then calculated from the three replicates of each treatment. A decolorized zone appeared when the fungus degraded the dye. Media phs were adjusted to 5.4 by using either highly diluted HCl or NaOH. Statistical analysis The data presented are the averages of the results of three replicates with a standard error of less than 5%. RESULTS AND DISCUSSION Control group Organisms used in this study were incubated as described in Material and Methods at 26 o C for at least 16 days. During this part of the study, no dye was added into the basal medium in order to compare the growth rates of each organism used in this study. As depicted in Fig. 1, P. citrinopileatus and P. eryngii are relatively slow growing organisms compared to the other types used in this study. Although P. djamor, P. ostreatus and P. sajor-caju completed their growth in 1 days, it took at least another 4 days for P. citrinopileatus and P. eryngii to cover the Petri-dishes completely. Among all, P. citrinopileatus was the slowest growing organism. Diameter of Growth (mm) Incubation Time (days) P. citrinopileatus P. djamor P. eryngii P. ostreatus P. sajor-caju FIGURE 1 - Radial growth rate of control group (dye conc.= mg L -1 ; using only Kirks s basal medium and 1 g L -1 glucose; T=26 o C). 131

3 by PSP Volume 16 No Fresenius Environmental Bulletin Indanthren Blue Dye Diameter of Growth at Day 12 (mm) 2 mg l-1 2 mg l-1 5 mg l-1 2 mg l-1 mg l-1 Remazol Blue dye Diameter of Growth at Day 12 (mm) 2 Organism mg l-1 2 mg l-1 5 mg l-1 2 mg l-1 mg l-1 Indanthren Yellow F3 Collosiol Diameter of Growth at Day 12 (mm) 2 Organism mg l-1 2 mg l-1 5 mg l-1 2 mg l-1 mg l-1 Blu CC Dranix Diameter of Growth at Day 12 (mm) 2 Organism mg l-1 2 mg l-1 5 mg l-1 2 mg l-1 mg l-1 Levafix Diameter of Growth at Day 12 (mm) 2 Organism mg l-1 5 mg l-1 1 mg l-1 2 mg l-1 5 mg l-1 FIGURE 2 - The effect of dye type and concentration on the growth of organisms studied. 1311

4 by PSP Volume 16 No Fresenius Environmental Bulletin Organism TABLE 1 - Agar-plate screening for decolorisation and radial growth inhibition. Decolorisation A Po Pe Pd Ps Pc Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye IB ± c ± c ± c ± c ± c RB LBB IYFC ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d ± d BCCD Organism Radial Growth B Po Pe Pd Ps Pc Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye Conc.(mg L -1 ) Dye IB RB LBB IYFC BCCD A : + visible decolorisation occurred; ++ full decolorisation; - no decolorisation; ± turned into another color; c : turned into yellow d : turned into brown; B : + better growth; - retarded growth; -- growth inhibition; Po: P. ostreatus; Pe: P. eryngii; Pd: P. djamor; Ps: P. sajor-caju; Pc: P. citrinopileatus; IB: Indanthren Blue; RB: Remazol Brilliant Blue; LBB: Levafix Brilliant Blue; IYFC: Indanthren Yellow F3GC Collosiol; BCCD: Blue CC Dranix. Effect of dye type and concentration on the growth of organisms studied The effects of dye type and concentration on the growth of organisms studied are depicted in Fig. 2. The diameter of radial growth value at day 12 was chosen for this purpose and reported in Fig. 2. Table 1 also shades light on the growth of organisms studied under the effect of increasing concentrations of dyestuff used in this study. Indanthren Blue CLF Colloisol (IB) IB did not result in remarkable growth inhibition for all organisms studied. On the contrary, better growth of organisms compared to control was observed in most cases indicating that they used the dye as substrate. As the dye concentration increased, the mycelium growth rate decreased accordingly, but not less than control group. Nonetheless, no decolorisation in all agar plates was observed, except for the experiments with dye concentrations of less than mg L -1, when color turned into yellowish-brown. While P. djamor, P. ostreatus and P. sajor-caju organisms spread out over 1 mm in diameter on the second day, P. eryngii and P. citrinopileatus did not. If the results are evaluated organism by organism, it can be seen that P. citrinopileatus completed its growth (coverage of agar plate; 9 mm diameter) on the 12 th day of experiments for all dye concentrations used, except for mg L -1. For control groups, which were not fed by any dye, the coverage of the agar plate was completed after 16 days. As the dye concentration increased up to 2 mg L -1, a slight decrease in growth was noted (4-5 mm less growth), but this was more pronounced for dye concentrations of mg L -1. For dye levels up to mg L -1 (not shown here), P. djamor presented a similar growth pattern as the control group (no dye used), and it was not observed any negative effect of dye. Although dye concentration over mg L -1 resulted in growth retardation (2 days), P. djamor was able to cover the agar plate completely. It is obvious that there was no inhibitory effect of IB up to mg L -1 and the organism was able to use the dye as substrate, similar to P. eryngii, P. ostreatus and P. sajor-caju organisms, resulting in better mycelium growth pattern compared to the control group. Indanthren Yellow F3GC Collosiol (IYFC) All organisms exposed to IYFC dye were able to show a complete growth on the agar plates at all dye concentrations studied. A slight retardation in mycelium growth was observed for P. eryngii, P. ostreatus and P. sajor-caju groups at mg L -1. Although there was no significant inhibition in growth, the color of the dye turned into brown increasing in darkness with dye concentration used. While the control group organisms covered the agar plate within 16 days, it took only 12 days when they were fed with less than mg L -1. P. eryngii was the slowest growing organism throughout these studies. P. ostreatus and P. sajor-caju were more active and faster growing organisms among the others. They almost completed their growth in less than 7 days. Blue CC Dranix (BCCD) BCCD with concentrations of over 2 mg L -1 resulted in retarded growth of all organisms, especially pronounced in experiments using P. eryngii and P. citrinopileatus. The 1312

5 by PSP Volume 16 No Fresenius Environmental Bulletin growth of P. citrinopileatus was completely hindered at mg L -1 BCCD. Even at the 32 nd day of the experiment, there was no sign of growth for P. citrinopileatus on the agar plate. The similar inhibitory effect of 2 mg L -1 dose was observed during the first 8 days of the experiment. Afterwards, P. citrinopileatus organism started to acclimate and grow with significantly low growth rates compared to control group, never being able to cover the whole plate at the end of the study (32 nd day). On the other hand, lower concentrations of this dye were consumed as substrate with no problem by P. citrinopileatus and the rest of the organisms studied, and growth rates for lower concentrations of the dye were higher compared to the control group. Each experiment was carried out in triplicate, but there was no decolorisation at all. P. djamor showed a gradual increase in growth up to the 5 mg L -1 dye, but at higher concentrations of the dye, again the growth rate decreased compared to the control group. P. djamor organism covered over 5% of the whole plate in about 6 days for concentrations lower than 2 mg L -1. With increasing dye levels (2 and mg l -1 ), the time to cover the plate decreased accordingly, and full growth was observed at the 12 th and 14 th day of the experiment for agar plates using 2 and mg L -1, respectively. P. eryngii showed almost no growth during the first days of the experiment, but, later on, a gradual increase in growth, especially for dye concentrations lower than 2 mg L -1 was observed. The initial growth rate for 2 and mg L -1 was significantly low for the first 1 days. It showed a significant adaptation after the 12 th day, especially for dye concentration of 2 mg L -1. P. eryngii organism exposed to mg L -1 did grow only 45 mm in diameter, at the end of 32 nd day. P. ostreatus and P. sajor-caju had faster growth patterns compared to the other organisms examined, and dyes applied had almost no negative effect on physiology of both organisms used. Only at mg L -1, a slight decrease in growth rate was observed, but both were still able to cover the whole plate in less than 12 days. Remazol Brilliant Blue BB (RB) RB was one of the dyes that was partially or completely decolorised by Pleurotus organisms used, and extent of decolorisation was a function of dye concentration. Due to the acidic nature of RB (following addition of this dye, agar medium ph dropped down to 4.5), before inoculating the agar plates, ph was adjusted to 5.5 using diluted NaOH. All white-rot fungi used in this study resulted in varying degree of decolorisation at dye concentrations less than 3 mg L -1. There was no problem in growth for all organisms, except for the experiments with mg L -1, which inhibited the growth of P. djamor and P. citrinopileatus. None of the organisms studied, except for P. ostreatus and P. sajor-caju, showed any decolorisation during the first 6 days of experimental period. For the first 8 days, P. citrinopileatus was not able to grow at dye concentrations of, 2 and mg L -1, respectively. From the 1 th day, this organism adapted itself and started to spread its mycelium, indicating a good acclimation potential. This acclimation potential was even more obvious at dye concentrations of mg L -1. A slight decolorisation was observed for the experimental set-up using 2 mg L -1, after 2 th day of incubation. A significant inhibition in growth was observed for P. djamor at dye concentrations over mg L -1. Even at day 28, nearly 5% of the agar surface was not covered by the mycelium of this organism. Decolorisation starting from day 24 was observed at dye levels less than mg L -1. P. eryngii showed a faster growth compared to the other fungi, and also its degree of decolorisation was twofold higher. Decolorisation started at day 6, and became quite visible at day 8 for dye concentrations of less than mg L -1. The results of the 2 and mg dye concentration experiments were interesting, because after day 2, a significant increase in mycelium growth was observed indicating the acclimation capability of the fungi. P. ostreatus and P. sajor-caju showed the most excellent growth and decolorisation capability. Parallel to their fast growth, they started to decolorise the media after 6 days, but for dye levels higher than 5 mg L -1, decolorisation degree was reduced. Levafix Brilliant Blue EB (LBB) LBB was the most inhibitory dyestuff used in this study, resulting in growth inhibition of all organisms at dye concentrations over 2 mg L -1. At lower LBB levels, a significant decolorisation was observed. Decolorisation Results During the decolorisation experiments, the following dyes were used: IYFC, BCCD, IB, RB and LBB at levels varying between 2 mg L -1, except for LBB, which was growth inhibiting for all organisms at concentrations over 2 mg L -1 (Table 1). As summarized in Table 1, RB and LBB were the only dyes being decolorized by all organism types used. In the experiments using IB and IYFC, color turned into yellow and brown, respectively. Even though no inhibitory effect of BCCD on mycelial growth was noted, none of the fungi was able to decolorize it in any of the experiments. CONCLUSIONS By far, white rot-fungi represent the single class of microorganisms most efficiently breaking down synthetic dyes [11, 12]. Especially white-rot fungi containing Pleurotus species are robust, and generally more tolerant to high concentrations of polluting chemicals than bacteria [7, 8]. They constitute a diverse ecophysiological group comprising mostly basidiomycetous fungi, capable of extensive aerobic lignin depolymerization and mineralization. This 1313

6 by PSP Volume 16 No Fresenius Environmental Bulletin property is claimed to be based on the white-rot fungi s capacity to produce one or more extracellular lignin-modifying enzymes [12]. Fungal decolorisation is a promising alternative to replace or supplement present treatment processes. Dye molecules have many different and complicated structures influencing fungal decolorisation, which is also a function of dye type. White rot fungi are the only organisms which can more efficiently degrade polymeric components to monomeric subunits. They are also unique organisms, which are directly responsible for the oxidative depolymerization of aromatic macromolecules. A fungus capable of decolorising one dye has different capacities for other dyes. There is a need to develop fungal strains which are capable of decolorising dye wastewater. In this study, it was shown that low dye concentrations (<2 mg L -1 ), except for LBB dye resulting in growth inhibition even at 2 mg L -1, were well tolerated by the organisms used. RB and LBB were the only dyes decolorised by almost all organisms, indicating that further research is needed to develop a practical application. This type of preliminary screening studies is helpful to reveal the white rot fungi and dyestuff interactions. Thereby, potential candidates for the biological decolorisation of each dye could be scaled up for field applications. [6] Fu, Y. and Viraraghavan, T. (21) Fungal decolorization of dye wastewaters. Bioresource Technology, 79, [7] Hou, H., Zhou, J., Wang, J., Du, C. and Yan, B. (24) Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochemistry, 39(11), [8] Levin, L., Papinutti, L. and Forchiassin, F. (24) Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes. Bioresource Technology, 94, [9] Yonni, F., Moreira, M.T., Fasoli, H., Grandi, L. and Cabral, D. (24) Simple and easy method for the determination of fungal growth and decolourative capacity in solid media. International Biodeterioration & Biodegradation, 54(4), [1] Weitz, J.H., Ballard, A.L., Campbell, C.D. and Killham, K. (21) The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi. FEMS Microbiology Letters, 22(2), [11] Balan, D.S.L. and Monteiro, R.T.R. (21) Decolorization of textile indigo dye by ligninolytic fungi. Journal of Biotechnology, 89, [12] Wesenberg, D., Kyriakides, I. and Agathos, S.P. (23) Whiterot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnology Advances, 22, ACKNOWLEDGEMENTS The authors wish to thank TUBITAK-CAYDAG for the financial support of this study under the grant No. 14Y393. The data presented in this article were produced within the projects above. However, only the authors of this article are responsible for the results and discussions made herein. REFERENCES [1] Galindo, C. and Kalt, T. (1999) UV/H 2 O 2 oxidation of azodyes in aqueous media: evidence of a structure degradability relationship. Dyes and Pigments, 42(3), [2] Robinson, T., McMullan, G., Marchant, R. and Nigam, P. (21) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresource Technology, 77, [3] Azbar, N., Yonar, T. and Kestioglu, K. (24) Comparison of various advanced oxidation processes and chemical treatment methods for COD and color removal from a polyester and acetate fiber dyeing effluent. Chemosphere, 55, [4] Kapdan, K. and Kargi, F. (22) Simultaneous biodegradation and adsorption of textile dyestuff in an activated sludge unit. Process Biochemistry, 37, [5] Gottlieb, A., Shaw, C., Smith, A., Wheatley, A. and Forsythe, S. (23) The toxicity of textile reactive azo dyes after hydrolysis and decolourisation. Journal of Biotechnology, 11, Received: December 5, 26 Revised: February 5, 27 Accepted: May 2, 27 CORRESPONDING AUTHOR Erbil Kalmış Ege University Faculty of Engineering Department of Bioengineering 35 Bornova, Izmir TURKEY erbil.kalmis@ege.edu.tr FEB/ Vol 16/ No 1/ 27 pages