Decolorization of Six Synthetic Dyes by Fungi

Corresponding Author: Mika A. Kähkönen Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, P.O. Box 56, Biocenter 1, FI 00014 University of Helsinki, Finland Email: mika.kahkonen@helsinki.fi Abstract: To find out ability of fourteen basidiomycetes and four ascomycetes strains to grow in the presence of synthetic colour dyes and to degrade them, fungi were cultivated on the malt agar plates containing 0.5 g kg −1 dye, either Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B or Reactive Black 5. Fungi representing basidiomycetes were Phlebia radiata (FBCC 43), Tremella encephala (FBCC 1145), Dichomitus squalens (FBCC 312), Physisporinus rivulosus (syn. Obba rivulosa, FBCC 939), Cerrena unicolor (FBCC 387), Pleurotus abieticola (FBCC 517), Phanerochaete velutina (FBCC 941), Agrocybe praecox (FBCC 476), Trametes pubescens (FBCC 735), Pleurotus ostreatus (FBCC 498), Fomitopsis pinicola (FBCC 18), Postia placenta (= syn. Rhodonia placenta, FBCC 112), Gloeophyllum trabeum (FBCC 328) and Piptoporus betulinus (FBCC 1191). Ascomycetes belonged to genera Alternaria (HAMBI 3289), Epicoccum (HAMBI 3291), Fusarium (HAMBI 3292) or Chaetomium (HAMBI 3291). The growth rate of P. rivulosus belonged to three highest among the 14 tested basidiomycetes with five dyes, but not in the case of Remazol Brilliant Red F3B containing plates. The growth rate of A. praecox belonged to three lowest among the 14 tested basidiomycetes on Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Red F3B, Reactive Black 5 and malt agar. The growth rate of Chaetomium sp. was the highest among the four ascomycetes on all tested plates. Decolorization was seen with 7 basidiomycetous strains on Remazol Brilliant Blue R, with 7 basidiomycetes on Remazol Brilliant Orange 3 R, with 8 basidiomycetes on Reactive Blue 4 and 11 basidiomycetes on Reactive Black 5 containing plates. T. encephala did not decolorize any of the tested six dyes. Epicoccum sp. and Chaetomium sp. decolorized Reactive Black 5 dye containing plates. None of the fourteen basidiomycetous or four ascomycetes were able to degrade all the tested six dyes.


Introduction
Synthetic dyes are widely used in mass production of textiles, food and paper (Pandey et al., 2007). The synthetic dyes have complex chemical structures, which make them persistent against light, water and microbial attack (Saratale et al., 2011). This persistence makes them harmful in the environment. Considerable amounts of synthetic dyes have been released from industrial production and discarded products to the environment.
Ligninolytic basidiomycetous fungi produce different combinations of extracellular oxidoreductive enzymes (laccases, manganese peroxidases, versatile peroxidases and lignin peroxidases), which can degrade natural biopolymers such as lignin and degrade recalcitrant xenobiotic synthetic compounds (Hatakka, 1994;Hatakka and Hammel, 2010). The growth of ascomycetes and basidiomycetes and their extracellular enzyme production are suitable indicators for the environmental health. Synthetic colour dye compounds can decrease the growth of fungi and production of extra-and intracellular enzymes, which are needed in biodegradation of xenobiotic synthetic dyes. Some ascomycetes are able to produce extracellular oxidative enzymes (Palonen et al., 2003;Sánchez, 2009). Ascomycetes produce mainly hydrolytic enzymes, which degrade carbohydrates (Hatakka and Hammel, 2010;Rodriguez et al., 1996;Kluczek-Turpeinen et al., 2003). Because the variety of different dyes is enormous, only little is known about capabilities of lignocellulose degrading ascomycetes (molds) while the capabilities of lignin degrading wood and litter decomposing basidiomycetes to degrade synthetic dyes have gained interest during last decades (Wesenberg et al., 2003, Rodriguez Couto, 2009. Especially fungal laccases have been studied in larger screenings for bleaching of synthetic dyes.
The aim of the study was to assess the ability of taxonomically and functionally different fungi to grow in the presence of synthetic dyes and to degrade them as indicated by decolorization. We selected fourteen basidiomycetes and four ascomycetes.

Materials and Methods
The four litter-decomposing saprotrophic ascomycetes from different genera were an Alternaria sp. (

Colour Dye Plate Tests
The ability of fungi to grow and biodegrade synthetic colour dyes were tested with six different dyes. These were Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B and Reactive Black 5. 0.5 g dm −3 of these colour dyes were added on malt agar plates. The control was malt agar plate without added colour dyes. ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid, Sigma-Aldrich, U.S.A.) was used as indicator dye in the malt agar plate to test ability of fungi to produce laccase and other oxidative enzymes. The formation of green colour in the ABTS plate indicates the production of oxidative enzyme(s). The decolorization of the other five dyes indicates degradation of the tested dye compound. A 4 mm diameter fully grown agar plug from each tested fungus was added to the center of the test plate. All was done as triplicate. Plates were incubated at 25°C. The diameter of the growth and colour change were measured on each sector of 90° and averaged over the four sectors of the plate.

Results
The growth rates of 14 basidiomycetes were tested with Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B, Reactive Black 5 and ABTS and without added synthetic dyes on malt agar plates (Fig. 1). All these fourteen fungi, namely P. radiata, T. encephala, D. squalens, P. rivulosus, C. unicolor, P. abieticola, P. velutina, A. praecox, T. pubescens, P. ostreatus, F. pinicola, P. placenta, G. trabeum and P. betulinus grew in the presence of all the six selected synthetic dyes and ABTS in the plates. The growth rate of P. rivulosus was the highest among the 14 tested basidiomycetes on five different dyes, ABTS plates and malt agar plates, but it was not the case on Remazol Brilliant Red F3B containing plates. The growth rate of P. radiata belonged to three highest among 14 tested basidiomycetes on five different colour dye and malt agar plates, but this was not the case on ABTS or Remazol Brilliant Yellow GL containing plates. The growth rate of A. praecox belonged to three weakest among the 14 tested basidiomycetes on Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Red F3B, Reactive Black 5 and on malt extract agar without added dye.  Eleven of the tested basidiomycetes, namely the white-rot fungi P. radiata, D. squalens, P. rivulosus, C. unicolor, P. abieticola, P. velutina, T. pubescens and P. ostreatus, the litter-decomposing fungus A. praecox and the brown-rot fungi F. pinicola and P. placenta formed colour zone in ABTS plates (Fig. 3) indicating production of extracellular oxidative enzymes, either peroxidases or laccases. The colour zone formation was not seen on ABTS containing plates with T. encephala, G. trabeum and P. betulinus indicating that these fungi did not produce extracellular oxidative enzymes. Tremella encephala is a fungal parasite and G. trabeum and P. betulinus are brown-rot fungi.
The ability of four ascomycetes, Alternaria sp., Chaetomium sp., Epicoccum sp. and Fusarium sp., was tested to decolorize Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B, Reactive Black 5 or the formation of colour zone with ABTS containing malt agar plates. The formation of colour zone was seen with Alternaria sp., Epicoccum sp. and Fusarium sp. on ABTS plates indicating production of extracellular oxidative enzyme(s). The formation of colour zone was not seen with Chaetomium sp. on ABTS plates. Decolorization was not seen with any of tested six colour dyes with Alternaria sp. Decolorization was seen with Epicoccum sp. and Chaetomium sp. on Reactive Black 5 dye plates. Decolorization was seen with Fusarium sp. on Reactive Black 5 dye plates.

Discussion
Our results show that anthraquinone dye Remazol Brilliant Blue R, azo dye Remazol Brilliant Orange 3 R, anthraquinone dye Reactive Blue 4, azo dye Remazol Brilliant Red F3B and azo dye Reactive Black 5 were decolorized by some of the 14 tested basidiomycetes.
None of the tested fungi was able to decolorize all the synthetic dyes in our experiments. Azo dye Remazol Brilliant Yellow GL was not decolorized by any of the 14 tested basidiomycetes. P. ostreatus decolorized well Drimarene Orange K-GL, Procion BluePX-5R and Cibacron Blue P-3RGR in liquid culture during 10 days except Remazol Brilliant Yellow (Asgher et al., 2006). Our results and the results of Asgher et al. (2006) indicate that Remazol Brilliant Yellow GL is persistent in the environment.
Remazol Brilliant Orange 3 R, Reactive Blue 4 and Reactive Black 5 dye were decolorized by D. squalens, P. rivulosus, P. velutina, A. praecox and T. pubescens indicating that these fungi are potentially useful for biodegradation and bioremediation in multi contaminated soils. Our results showed that Remazol Brilliant Red F3B was decolorized by P. radiata, P. rivulosus and T. pubescens indicating that they could be useful in the treatment of soil or water contaminated by this dye. Earlier it was found that the white-rot fungi Bjerkandera sp., Phanerochaete chrysosporium, T. hirsuta and T. versicolor are able to decolorize Remazol Black B, Reactive Blue 15 and Remazol Brilliant Orange in solid plate during 10 days (Swamy and Ramzay, 1999).
Our results from ABTS plate tests indicate that the white-rot fungi P. pulmonarius, P. radiata, D. squalens, P. rivulosus, C. unicolor, P. abieticola, P. velutina, T. pubescens, P. ostreatus, the litter-decomposing fungus A. praecox and the brown-rot fungi F. pinicola and P. placenta produced laccase or other oxidative enzymes. T. encephala, G. trabeum and P. betulinus did not produce any extracellular oxidative enzyme (laccase) under our conditions. Recent comparisons of basidiomycete genomes show that the borderline between white-rot and brown-rot fungi is not as strict as previously thought and many brown-rotters such as F. pinicola, P. placenta and G. trabeum do have laccase genes in their genomes (Riley et al., 2014). Laccase is produced by D. squalensis (Susla et al., 2007), P. rivulosus (Hakala et al., 2005), T. pubescens (Palmieri et al., 2000), F. pinicola (Gramss et al., 1999), P. placenta (Wei et al., 2010), P. pulmonarius (De Souza et al., 2004), P. radiata (Arora and Gill, 2001), P. abieticola (Koutrotsios and Zervakis, 2014), P. velutina (Boddy, 2000), A. praecox and P. ostreatus . Although the studied basidiomycetes P. pulmonarius, P. radiata, D. squalens, P. rivulosus, C. unicolor, P. abieticola, P. velutina, A. praecox, T. pubescens, P. ostreatus, F. pinicola and P. placenta produced laccase, they did not decolorize all studied dyes indicating that other enzymes than laccases were involved in decolorization of the tested dyes. Lignin degrading peroxidases (PODs, Class II peroxidases) produced the white-rot fungi have certainly an important role in the degradation of dyes, but it does not explain the decolorization caused by brown-rot fungi because the most common brown-rot fungi do not produce Class II peroxidases, since there are no POD encoding genes in their genomes (Riley et al., 2014). All fourteen tested basidiomycetes were able to grow in the presence of all tested six dyes and the redox indicator ABTS. The growth rate of P. rivulosus and P. radiata belonged to three highest and the growth rate of A. praecox to three lowest among the 14 tested basidiomycetes on Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Remazol Brilliant Red F3B, Reactive Black 5 and on the control malt extract plates without any added dyes. We have previously studied the effect of different metals on the growth of many fungi studied also in the present work. The growth of A. praecox was tolerant to Mn, Cr and Li (Hartikainen et al., 2013). The growth of P. radiata was almost completely inhibited with 2 mg Co/kg (63%) and with 100 mg Li/kg (88%) (Hartikainen et al., 2013). The growth of P. rivulosus was sensitive to Mn (50-400 mg kg −1 ), Cr (20-100 mg kg −1 ), Cd (5-10 mg kg −1 ), Li (20-100 mg kg −1 ) and Co (20 mg kg −1 ) (Hartikainen et al., 2013). The growth of P. radiata and P. rivulosus was sensitive to Al, Mo and W and tolerant to Zr. The growth was sensitive to Ga in the case of P. radiata and sensitive to V in the case of P. rivulosus (Kluczek-Turpeinen et al., 2014). The growth of the tested ascomycete Chaetomium sp. was sensitive to Cr, Co and Li and Alternaria sp. to Cd (Hartikainen et al., 2013). The growth of the ascomycetes Alternaria sp. and Fusarium sp., was sensitive to six metals (Al, Mo, V, Zr, W, Ga) (Kluczek-Turpeinen et al., 2014). Our results showed that P. radiata and P. rivulosus were fast growing, but the growth of these two fungi is sensitive to several metals (Hartikainen et al., 2013;Kluczek-Turpeinen et al., 2014) indicating that their ability to grow is vulnerable in the metal contaminated waste or soil. Our results indicate that P. rivulosus and P. radiata are tolerant and suitable for the fast bioremediation of the dyes; namely Remazol Brilliant Blue R, Remazol Brilliant Yellow GL, Remazol Brilliant Orange 3 R, Remazol Brilliant Red F3B, Reactive Black 5 and malt agar plates. The slow growth rate of A. praecox was not useful for efficient decolorization of the dyes. The growth rate of Chaetomium sp. was the highest among tested ascomycetes on all tested plates indicating that it was tolerant to all tested dyes. Basidiomycetes were generally more efficient to decolorize dyes than ascomycetes.

Conclusion
All fourteen selected basidiomycetous fungi P. radiata, T. encephala, D. squalens, P. rivulosus, C. unicolor, P. abieticola, P. velutina, A. praecox, T. pubescens, P. ostreatus, F. pinicola, P. placenta, G. trabeum and P. betulinus grew in the presence of all six selected synthetic dyes and ABTS in the plates. The growth rate of P. rivulosus belonged to three highest among the tested basidiomycetes with five dyes, Remazol Brilliant Blue R, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B and Reactive Black 5. The growth rate of Chaetomium sp. was the highest among four ascomycetes on all tested plates. The fast growth rate is beneficial in bioremediation of synthetic dye contaminated environment. Remazol Brilliant Blue R, Remazol Brilliant Orange 3 R, Reactive Blue 4, Remazol Brilliant Red F3B and Reactive Black 5 were decolorized with 14 tested basidiomycetes. None of tested fungi was able to decolorize all tested synthetic dyes indicating that abilities of several fungi are needed in the bioremediation of multi contaminated sites. Remazol Brilliant Yellow GL was not decolorized by any of 14 tested basidiomycetes indicating that it is a very persistent dye in the environment.