Screening of Fungi for Decolorization of Dye Wastewater

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1 International Proceedings of Chemical, Biological and Environmental Engineering, V0l. 100 (2017) DOI: /IPCBEE V Screening of Fungi for Decolorization of Dye Wastewater Merih Kıvanc +, Mine Doğruer Özen Anadolu University, Faculty of Science,Department of Biology, Eskişehir, TURKEY Abstract. A total of 40 fungi were screened for their ability to decolorize Xiron orange RHD (FW), Tobactive scarlet P2R (Kimsa). Microorganisms having the ability of decolourization of organic colorants were isolated from Porsuk stream, soil and wastewater of textile factory. Four strains of Fusarium, Penicillim expansum, P. citreo-viride, Aspergillus flavipes, Trichoderma harzianum Paecilomyes variotii have been chosen for this study. In addition to these strains four different strains of Myrothecium were also used in this study. By using the those strains, maximum decolourisation was observed at 25oC ph 7.0.Among these, maximum decolourisation was obtained by A. flavipes. It was followed by T. harzianum, Fusarium sp2. These strains has decreased B.O.D. degree of was the wastewater of textile factory on the rates of 17.7% and 24.2%. Keywords: texile dye, fungi, decolourization 1. Introduction Azo compounds are used extensively in the food, pharmaceutical, cosmetic and textile industries [1]. Aromatic azo groups are not synthesized in nature, azo dyes are considered to be xenobiotics [2]. As consequence azo dyes are recalcitrant in aerobic wastewater treatment plants. However, provided that the proper conditions and microorganisms are used, biodegrading of azo dyes is possible [3]. Wastewater treatment systems generally do not remove the dyes, wastewaters from textile industry result in pollution of the environment. The elimination of collared effluents in wastewater is based mainly on physical or chemical methods. Although these methods are effective, they suffer from such shortcomings as high cost, formation of hazardous by-products and intensive energy requirements. Therefore, as a better alternative, microbial biodegradation methods are receiving attention. The use of white-rot fungi has attracted increasing attention as these organisms have the ability to metabolize a diverse range of polluting compounds. Phanerochaete chrysosporium, the most extensively studied white-rot fungus, has been shown to metabolize and decolorize textile dyes [4], [5]. Bio-decolourization of lignin-containing pulp and paper wastewater using white rot fungi P.chrysosporium and Tictoporia sp. Due to high oxidative potential of many of the enzymes associated with white rot fungi, e.g. ligninase, laccase, Mn-peroxidase [6], [7]. Several other dye decolorizing fungal species have been reported, which include Aspergillus niveus 2 ve Fusarium moniliforme [8]. It is thus not surprising those efforts to isolate from nature microorganisms utilizing azo dyes as carbon sources where unsuccessful. However, adaptation experiments in chemostats and carefully adjusted selective pressure let to bacterial cultures which mineralised the carboxylated azo dyes. As for dye colour removal, review [9], [10] described the ability of Rhodococcus, Bacillus cereus and Plasmiomonas/Achromobacter to degrade soluble dyes, acid red dye and five azo-dyes, respectively. On the other hand, textile dyes were found strongly adsorbed and held by wastewater treatment plant sludge that was land filled. This suggests that adsorption may play another key role in bio- decolourization. Corresponding author. Tel.: address: mkivanc@anadolu.edu.tr 1

2 There is not much information about the effects of thenon-white-rot fungi on decolourization of azo dyes. In this research, isolated fungi were used and the ability of these organisms to decolorize Xiron orange RHD(FW),Tobactive scarlet P2R(Kimsa)tested. 2. Materials And Methods 2.1. Microorganisms Myrothecium leutricum, M.penicilloides, M.masonii obtained from Norten Research Center, USA, Peoria IL. Fusarium sp.1, Fusarium sp2, Fusarium sp 3, Fusarium sp4, Penicillium expansum Aspergillus flavipes, P.sitreo-viride, Tricoderma harzianum and Paesilomises variotti was isolated in our laboratory. They were maintained through periodic transfers on sabauroud dextrose agar at +4 o C. Subculters were made every 3 to 4 weeks Dyes Xiron orange RHD (FW), Tobactive scarlet P2R (Kimsa) were obtained from textile fabric, Eskişehir, Turkey Wastewater A textil dye factory in Eskişehir provided the wastewater. From a textile dyeing factory, Sample I wastewater from dyeing, ph 7.5 dark green Sample II wastewater from dyeing, ph 7.0 Sample III wastewater from Sample IV wastewater from Sample V wastewater from dyeing, ph 8.5 brown, flom dyeing, ph 7.5 dark dyeing, ph 8.5 Lila 2.4. Isolation of Fungi Water samples were collected from Porsuk river in Eskişehir (Turkey) that is heavily polluted by textile wastewater. And soil samples were collected textile fabric in Eskişehir. Nutrient agar and potato dextrose agar petri plates supplemented with dyes (1%) used to screen soil, water and waste water samples for colonies circlet by a clear decolorized zone. Isolates were identified by using the methods and identification keys for fungi (Hasenekoğlu 1991). These fungi were then tested for dye decolourization under submerged culture condition at near ambient temperature for up to two weeks Decolourisation of Dyes with Fungi Cultures were cultured at 25 o C in malt extract broth. After one week of incubation, they were filtered with Whatman no 1 filter paper then weighed. Wet cell cake were mixed with specific aqueous dye solution (0.1%) in 1:3 weight ratio and incubated for 1 and 2 week. OD measured after 1 and 2 week. Decolorizing activity of Tobactive scarlet P2R. Xiron orange RHD were assayed by measurement of the decrease in colour density at 663nm, 514nm and 490nm respectively. The decolorizing yield was expressed as the degree of the decrease in absorbance at the same wavelength. Each treatment was carried out in duplicate and results obtained are given as the arithmetic mean Effect of Dye Concentration The culture of Fusarium sp2, A.flavipes and T. harzianum was gradually exposed to increasing concentration of dye (0.1mg/l, 1mg/l,10mg/l). Decolorizing activity of Tobactive scarlet P2R, Xiron orange RHD were assayed by measurement of the decrease in colour density at 663nm and 514nm respectively Effect of ph 2

3 The ph of the individual culture was adjusted to 3.0, 5.0, 6.5 and 7.0 and all cultures were incubated at 25 o C.Decolorization of dyes were monitored. The absorbance was measured at 663nm and 514nm to determine the concentration of Tobactive scarlet P2R. Xiron orange RHD Effect of Temperature Individual cultures were incubated at 5, 25 and 35 o C. Decolorization of Xiron orange RHD (FW) (Orange 13), Tobactive scarlet P2R (Kimsa) (Red mix) were determined the cell free supernatant. Its absorbance was read at OD663 Tobactive scarlet P2R OD514 Xiron orange RHD OD490. Decolourization of textile dyes were determined as follows; Decolorization (%)=Initial absorbance- absorbance Initial absorbance 2.9. Decolorization of Dye Containing Wastewater by Fungi Real dye containing wastewater samples were added to fungi and its effectiveness in dye color removal evaluated. They were each mixed with 5 days old mycelia in roughly 2.5:1 weight ratio and observed after 1-2 weeks static. 3. Result And Discussion Xiron orange RHD (FW), Tobactive scarlet P2R (Kimsa) which is monoazo dye has seen extensive in textile dying. Fusarium sp., P.expansum, P. citreo-viride, A.flavipes, T. harzianum, P.variotii were capable of decolorizing Xiron orange RHD (FW), Tobactive scarlet P2R (Kimsa) produced clear zones surrounding its colonies on the agar plates. Mou et al [11] and Karaca ve Kıvanç [8] also reported similar results. Mou et al [11] reported the decolourization activity of the Myrothecium and Ganoderma culture filtrate. Maximal decolorizing activity of A. flavipes are within 14 days. Bio-decolourisation was evident and effective all turned colourless to naked eyes. T. harzianum also showed high decolorizing ability for Tobactive scarlet P2R. The Trichoderma species were degrade aromatic pollutants [12]. A. niger, F. oxysporum and Trichoderma lignorum were degrade textile dye [13]. The effects of the initial medium ph on the decolorizing activity are shown in Table 1 and Table 2. From these results it was determined that the best ph value for decolourization is 7.0 for fungi. This gives the advantage that it is not necessary to adjust the initial ph of this type of dye effluent. The maximum decolorizing activity for fungi was recorded within 2 weeks. M. masonii, M. cinctum, T. harzianum, Fusarium sp.2 and A. flavipes had a higher decolorizing activity than other test fungi. The effect of temperature on the decolorizing activity is shown in Table 3and Table 4. As seen from the tables, optimal temperature of decolorization for tested fungi is 25 o C..M. mansonii, T. harzianum and Fusarium sp2 shows higher decolourisation rate for Tobactive scarlet P2R than that of the other fungi. A. flavipes, M. leutricum and M.cinctum shows higher decolorization rate for Xiron orange RHD (FW) than that of the other fungi. High decolorizing activity was obtained within 2 weeks of incubation at 25 o C. Table 1. Decolorization of Tobactive Scarlet P2R by Fungi. ph Fungi 1w 2w 1w 2w 1w 2w 1w 2w Fusarium sp Fusarium sp Fusarium sp.3 _ Fusarium sp P.expansum A.flavipes P.citreo-viride T.harzianum P.variotii M.penicilloides M. masonii M.leutricum M.cinctum x100

4 Table 2. Decolorization of Xiron Orange RHD by Fungi ph Fungi 1w 2w 1w 2w 1w 2w 1w 2w Fusarium sp Fusarium sp Fusarium sp.3 _ Fusarium sp P.expansum A.flavipes P.citreo-viride T.harzianum P.variotii M.penicilloides M. masonii M.leutricum M.cinctum Table 3. Effects of Temperature Decolorization of Tobactive Scarlet P2R Temperature( o C) Fungi 1w 2w 1w 2w 1w 2w Fusarium sp Fusarium sp.2 _ Fusarium sp Fusarium sp P.expansum A.flavipes _ P.citreo-viride T.harzianum P.variotii M.penicilloides M. masonii M.leutricum M.cinctum Table 4. Effects of Temperature Decolorization of Xiron Orange RHD Temperature( o C) Fungi 1w 2w 1w 2w 1w 2w Fusarium sp Fusarium sp Fusarium sp Fusarium sp P.expansum A.flavipes P.citreo-viride T.harzianum P.variotii M.penicilloides M. masonii M.leutricum M.cinctum The relationship between dye concentration and decolourizing activity are shown in Table 5. The decolorizing activity increased in parallel with the concentration size. Complete decolourisation (100% removal) of dye was not observed cultures. When 10mg/l of the dye was added to the medium, a maximum 4

5 decolourizing rate 59.1% -40.7% was obtained at 7 days. Earlier studies of biodegradation of azo dyes showed that these dyes are relatively resistant to microbial biodegradation in the environment [14]. These dyes are toxic to many microorganism. This study showed that decolourization can be obtained by using pellets of tested fungi. It has been determined that whole tested fungi were able to decolorize Xiron orange RHD (FW), Tobactive scarlet P2R(Kimsa). Control experiments done in the absence of fungal inoculum showed negligible colour loss from the medium, thereby ruling out the possibility of colour removal due to abiotic mechanisms. Results obtained by us compare favourably to those reported for strains of Myrothecium verrucaria and Gonoderma sp., whice are capable of decolourising effluent containing wide range of dyes mainly through adsorption to mycelium [11] (Mou et al. 1991). Zheng et. al. [15] reported the same observation that Poly R-478 was initially adsorbed onto the Penicillium mycelia. The decolourization and biodegradation of various dyes by crude extracellular filtrate were study [1], [16], [17]. Sumathi and Manju [18] have demonstrated 95% decolourization through adsorption azo reactive dye by using Aspergillus foetidus. Kim et.al. [19] did not observed decolourization of Reactive blue 5 dye. Our results are contradictory to those observed for fungi such as P.chrysosporium, which decolourize several dyes through the action of lignin peroxidase and manganese peroxidese or M. verrucaria which remove colour trough cell adsorption processes [1]. The lignins degrading white rot fungi mineralize a wide variety of structurally diverse environmental pollutants. Due to the high oxidative potential of many of the enzymes associated with white rot fungi, they have been shown to exert a positive effect upon many environmental pollutants [1], [20]. Table 5. Effects of Temperature Decolorization of Dyes Concentration. Concentration 10mg/l 1 mg/l 0.1 mg/l Dye Fungi Tobactive scarlet P2R Xiron orange RHD Fusarium sp2 A. flavipes T. harzianum Fusarium sp2 A. flavipes T harzianum All of the tested fungi showed high degree of decolourisation on wastewater samples I, II,IV and V (Table VI) In wastewater sample III, after 1week of incubation the absorbances were approximately 50%, 40%, 48% of that initially present for A.flavipes, Fusarium sp 2, T. harzianum respectively. Although in all cases decolourisation values at the end of the incubation period were close to 40%, these were very different at week 2 of the experiment. In the sample I, a colour reduction of (% was already reached at week 1 of fungal treatment (A. flavipes and T. harzianum). On the other hand, a minimal colour decrease was observed sample III. The highest decolourisation values were achieved by A. flavipes in the Sample II. The biological oxygen demand results followed a similar tendency to those of the colour units decrease. In the same way, the final BOD reduction observed in all samples (Table 7). The dyes were decolorized to a greater extend by A.flavipes, Fusarium sp 2, T. harzianum than other fungi. This results show that higher rates of decolourisation can be obtained by carefully selecting the fungi. The time taken for tested fungi to decolourise these synthetic dyes compares favourably with reports for other fungi which indicate periods of between 7 and 20 days to achieve greater than 90% decolourise of diverse synthetic dye. [21], [22]. Geotricum candidum Dec.1 isolated from soil and capable of decolourising a number of antraquione dyes [19]. 5

6 Table 6. Decolorization (%) of textile dye containing effluents. Fungi A.flavipes Fusarium sp 2 T. harzianum Wastewater sample 1w 2w 1w 2w 1w 2w Sample I Sample II Sample III Sample IV Sample V Table 7. Decolourisation of textile dye containing effluents. BOI5(mg/l) BOI 5(mg/l) Wastewater A.flavipes Fusarium sp 2 T. harzianum Wastewater sample Sample I Sample II Sample III Sample IV Sample V There is need for development of novel biotreatment processes to ensure environmental protection from potential pollutants, either through complete mineralization via aerobic and sequential anaerobic-aerobic treatment processes or efficient bioadsorption from using microorganisms. We showed that the Xiron orange RHD(FW),Tobactive scarlet P2R were decolourized by isolated fungi Fusarium sp, P.expansum, A.flavipes, P.citreo-viride, T.harzianum, P.variotti. As a dye decolorizing fungus,phanerochaete chrysosporium has been extensively studied. Fusarium sp, A.flavipes, T.harzianum has advantages over P. chrysosporium is less sensitive to shear stress and to higher dye concentrations, and it occurs widely in soil and water. These results have suggested the potential of the pellet of fungi in bioremediation of dye contaminated water and waste water. Further studies are required to understand the decolourization mechanisms including adsorption/desorption kinetics and mineralization process of polymeric dyes by selected fungi. 4. Acknowledgements The financial support of the research foundation of Anadolu University is gratefully acknowledged. 5. References [1] C. Cripps, A. J. Bumbus and D. S. Aust, Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol.1990, 54: [2] T. Zimmermann, H. G. Kulla, T. Leisinge. Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur J. Biochem 1982, 129: [3] K. Wuhrmaann, K. Mencsner, T. Kappeler. Investigation on rate determining factors in the microbial reduction of azo dyes. European J Appl Microbiol Biotechnol. 1980, 9: [4] G. McMullan, C. Meehan, A. Conneely, N. Kirby, T. Robinson, P. Nigam, I. M. Banat, R. Marchant, W. F. Smyth Microbial decolourization and degradation of textile dyes. Appl. Microbiol Biotechnol.2001, 56: [5] Y. Fu and T. Viraraghavan.Fungal decolorization of dye wastewaters: a review. Bioreseource Technol. 2001, 3: [6] A. Paszczynski, B. M. Pasti-Grigsby, S. Goszczynski, L. D. Crawfort, L. R. Crawfort. New approach to improve degradation of recalcitrant azo dyes by Streptomyces spp. and Phanerochaete chrysosporium.enzyme Microb. Technol. 1991, 13:

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