Water Air Soil Pollut (2014) 225:2026 DOI 10.1007/s11270-014-2026-6 Comparative Analysis of Azo Dye Biodegradation by Aspergillus oryzae and Phanerochaete chrysosporium Graziely Cristina Santos & Carlos Renato Corso Received: 13 January 2014 / Accepted: 4 June 2014 # Springer International Publishing Switzerland 2014 Abstract The textile industry often releases effluents into the environment without proper treatment or complete dye removal. Azo dyes, which are characterized by azo groups (―N═N―), are frequently used in the textile industry. Among the different wastewater treatment methods available, biological treatment has been extensively studied. The aim of the present study was to compare the biodegradation of the azo dye Direct Blue 71 by the fungi Phanerochaete chrysosporium and Aspergillus oryzae in paramorphogenic form using a 100 μg/ml dye solution. Biodegradation tests were performed within 240 h. The absorbance values obtained with UV-VIS spectrophotometry were used to determine the absorbance ratio and the percentage of dye discoloration following the biodegradation test. FTIR analysis allowed the identification of molecular compounds in the solution before and after biodegradation. Both A. oryzae and P. chrysosporium demonstrated considerable potential regarding the biodegradation of dyes in wastewater. These results may contribute toward improving effluent treatment systems in the textile industry. G. C. Santos : C. R. Corso (*) Departamento de Bioquímica e Microbiologia, UNESP – Univ Estadual Paulista, Campus Rio Claro, Instituto de Biociências, Avenida 24 A, 1515 Bela Vista, 13506-900 Rio Claro, SP, Brazil e-mail: crcorso@rc.unesp.br G. C. Santos e-mail: grazielycs@gmail.com Keywords Direct Blue 71 . Fungi . Amines . Paramorphogenic form . UV-VIS . FTIR 1 Introduction Textile effluents contain substances from different stages of dyeing and finishing as well other processes. The pollutants found in these effluents are primarily persistent organic substances, such as dyes and salts, giving the effluent a low degree of biodegradability (Fu et al. 2011). Dyes are visible compounds that, when released into the environment, can cause the appearance of color in rivers, hindering the penetration of sunlight and thereby reducing the process of photosynthesis among different organisms in these ecosystems (Wang et al. 2005). Azo dyes are the class of dyes with the largest number of representatives, accounting for 60 to 70 %, and therefore constitute the majority of the components in effluents from the textile industry (Hunger 2003; Van der Zee et al. 2003). In addition to textile applications, this class of dyes is still widely used in pharmaceutical, food, and cosmetic industries. The main characteristic of azo dyes is the binding of aromatic rings by azo groups (―N═N―) which, added to sulfonated substitutions, contribute to the resistance of dyes to chemical and microbiological degradation processes (Martins et al. 2001; Stolz 2001; Hu and Wu 2001). There is a need to remove dye from waste effluents before it mixes with watercourses. Biological methods have typically been applied to remove organic compounds and color from textile effluents due to the low 2026, Page 2 of 11 cost and simple operation and maintenance of these methods (Hunger 2003). In an attempt to mitigate this contamination, studies have been conducted to evaluate the biodegradation of these compounds by microorganisms that have metabolic versatility and are capable of degrading structures such as those found in azo dyes. A large number of microorganisms belonging to different taxonomic groups have been reported to demonstrate the ability to decolorize azo dyes (Vitor and Corso 2008). Biodegradation occurs by enzymes that attack and break the most important chemical bonds in dyes (Mou et al. 1991). Fungi are widely used in biodegradation of azo dyes due to their main feature of producing extracellular enzymes able to degrade complex molecules. Species such as Aspergillus sp., Phanerochaete chrysosporium, Neurospora crassa, Rhizopus sp., and Pleurotus ostreatus have demonstrated considerable potential regarding the bioremediation of textile dyes (Corso and Almeida 2009; Kaushik and Malik 2009; Enayatzamir et al. 2010; Jesus et al. 2010; Teixeira et al. 2010; Corso et al. 2012). P. chrysosporium is the most widely studied white rot fungus in terms of the biodegradation of xenobiotic (Robinson et al. 2001). This microorganism has been used in the biodegradation of gaseous chlorobenzene (Wang et al. 2008), the pesticide endosulfan (Kullman and Matsumura 1996), the insecticide heptachlor (Arisoy and Kolankaya 1998), and a number of different dyes (Cripps et al. 1990; Paszczynski and Crawford 1995; Martins et al. 2001; Wesenberg et al. 2003; Santos et al. 2009). The ligninolytic system of this fungus is represented mainly by the enzymes laccase, lignin peroxidase, and manganese peroxidase, which are produced in media containing limited sources of carbon and nitrogen. These enzymes have the ability to depolymerize lignin and a variety of other compounds (Stolz 2001; Teixeira et al. 2010). The filamentous fungus Aspergillus oryzae is classified as an imperfect fungus (anamorphic) with no sexual stage in its life cycle (Galagan et al. 2005). This organism has been extensively used for the production of fermented foods and beverages. However, there is little information on its use for the biodegradation of pollutants, such as dyes. Thus, A. oryzae has been used with the aim of evaluating and comparing its biodegradation ability with that of P. chrysosporium, which is known for its efficiency in this process. Corso and Almeida (2009) evaluated the ability of A. oryzae regarding the bioremediation of dyes in textile effluents and identified its potential in removing azo dyes Water Air Soil Pollut (2014) 225:2026 from aqueous solutions. Shakeri et al. (2008) used a recombinant peroxidase from A. oryzae for the decolorization of the anthraquinone Remazol Brilliant Blue R dye. In the attempt to find alternatives for the treatment of textile effluents, the aim of the present study was to evaluate the biological treatment of the azo dye Direct Blue 71 using two filamentous fungi in paramorphogenic form, A. oryzae and P. chrysosporium, and compare the effectiveness of both. The paramorphogenic form was used to facilitate the quantification of the biomass needed for treatment. 2 Materials and Methods 2.1 Dye Direct Blue 71 (DB71), Color Index 34140 and CAS 4399-55-7, is a direct azo dye obtained from the SigmaAldrich Chemical Company, Inc. 2.2 Microorganisms and Culture Conditions P. chrysosporium (CCB 478) was obtained from the culture collection of the São Paulo Institute of Botany (Brazil), and A. oryzae (CCT 5321) was obtained from the culture collection of the André Tosello Tropical Research and Technology Foundation (Brazil). The microorganisms were kept in test tubes with a 2 % malt medium (Lodder 1970). The medium to P. chrysosporium culture was modified with the addition of 1 % peptone and 4 % glucose. Modified Minimum Mineral Medium (Pontecorvo et al. 1953), consisting of NaNO3, KH2PO4, KCl, MgSO4, 7H2O, glucose, yeast extract, and distilled water, was used for the growth of the mycelial pellets. P. chrysosporium and A. oryzae cultures were used for the paramorphogenic process after 7 days of culturing, following the method described by Marcanti-Contato et al. (1997). 2.3 Dye Biodegradation Test Samples were prepared in triplicate in test tubes with 1 ml of dye stock solution to 1,000 μg ml−1, 8 ml of distilled water at pH 2.5, adjusted with H2SO4 0.01 M, and 1.03 mg ml−1 (dry weight) of P. chrysosporium in paramorphogenic form. The tests with A. oryzae were prepared with 1 ml of stock solution of dye, 1.1 mg ml−1 Water Air Soil Pollut (2014) 225:2026 Page 3 of 11, 2026 (dry weight) of A. oryzae in paramorphogenic form, and 7.5 ml of distilled water at pH 2.5. The control was prepared without biomass with 9 ml of distilled water at pH 2.5 and 1 ml of dye stock solution. The samples were incubated at 30±1 °C, and scans were performed every 24 h in a UV-VIS spectrophotometer (Shimadzu UV-2401 PC), totaling 240 h at the end of the test. The scan occurred at wavelengths of 800 to 190 nm in quartz cuvettes with an optical path of 5 mm. The biodegradation of DB71 dye was analyzed from data obtained by UV-VIS spectrophotometry to determine the absorbance ratio, following the method described by Glenn and Gold (1983), as well as from data obtained by Fourier transform infrared spectroscopy (FTIR) by the change of the dye molecular structure before and after treatment. The samples of every triplicate were analyzed using the UV-VIS spectrophotometer and FTIR. Every spectrum was evaluated and the spectrum was chosen according the similarity between the results. control solution were dried for 48 h at 105±1 °C and remaining in a desiccator for 24 h for the manufacture of the disks. After drying, the disks were prepared with 1 mg of dry dye and 149 mg of KBr by compression to 40 kN for 5 min. The disks were scanned in the 400 to 4,000 cm−1 range, with 16 scans and 4 cm−1 resolution. Baselines were corrected to 4,000, 2,000, and 400 cm−1. The spectra were normalized and expressed in terms of absorbance. The Lorentzian deconvolution function was used for the analysis of overlapping bands, following the method described by Forato et al. (1998). Higher resolution methods are based on the separation of the peaks that make up the band of interest and the correlation of their intensities or areas with secondary structures. Deconvolution is a technique used to reduce the bandwidth of the spectra (Forato et al. 1998). 2.4 UV-VIS Spectrophotometry 3.1 Dye Biodegradation Test The absorbance ratio determined from data of UV-VIS spectrophotometry allows the identification of evidence of biodegradation, which was confirmed after the FTIR analysis of these solutions. The absorbance ratio values were determined by the ratio between absorbance at two different wavelengths (A583/A320), corresponding to the chromophore group (583 nm) and azo group (320 nm) (Silverstein et al. 1994). According to Glenn and Gold (1983), a compound shows signs of degradation when there is a great variation between the absorbance ratio value of the treatment and the absorbance ratio of the control. However, when the absorbance ratio remains constant, the predominant process is biosorption, i.e., absorbance decreases proportionally, with no change in the structure of the dye. 3.1.1 UV-VIS Spectrophotometry 2.5 Fourier Transform Infrared Analysis The analysis of the dye solution in an FTIR Shimadzu IRPrestige-21 spectrometer requires the preparation of potassium bromide (KBr) disks with dye. 2.6 Preparation of KBr Disks After each scan, samples from the degradation test were reserved for the manufacture of disks. The samples and 3 Results and Discussion Figure 2 shows the absorbance at the wavelengths of the chromophore (583 nm) and azo (320 nm) groups and the absorbance ratios for each 24 h over 240 h following treatment with P. chrysosporium (Fig. 1a) and A. oryzae (Fig. 1b). Variation occurred in the absorbance ratio of the treated samples in comparison to the control (0 h). According to Glenn and Gold (1983), the biodegradation occurs if the absorbance ratio values of the samples remain constant in this situation. Therefore, this variation suggests that the molecular structure of the dye has changed, possibly indicating degradation. For the P. chrysosporium, there was considerable variation in the absorbance ratio in the first 24 h of treatment, indicating the fungus was able to break bonds of the dye molecule within a short contact time (Fig. 1a). This fact was confirmed with analysis of the FTIR spectrum. Further changes occurred between 96 and 120 h, after which the absorbance ratio gradually decreased. Changes occurred in the first 48 h for the A. oryzae remaining relatively constant up to 144 h, when the absorbance ratio changed (Fig. 1b). Changes in the structure of the dye likely occurred at this time. 2026, Page 4 of 11 Water Air Soil Pollut (2014) 225:2026 Fig. 1 Absorbance values of chromophore and azo groups and absorbance ratio after treatment of DB71 dye with P. chrysosporium (a) and A. oryzae (b) Subpanels a and b of Fig. 2 display the absorption spectra of samples treated with P. chrysosporium and A. oryzae, respectively. The spectra correspond to the control, 24 and 240 h (representing the beginning and end of the treatment), and 144 h (representing an intermediate point of the treatment), when variations in the absorbance ratio occurred with both fungi. The absorbance at both 583 and 320 nm decreased with each scan, demonstrating the removal of the dye from the solution by the fungus. However, although the absorbance ratios provide data indicating the occurrence of biodegradation, this process can only be confirmed after the identification of the characteristic bands of each structure in the FTIR spectra. The absorption in UV-VIS allowed visualizing differences between the treatments with each fungus. After 24 h of treatment with P. chrysosporium, the dye demonstrated a different spectrum from the control, with the chromophore peak (583 nm) smaller and displaced. The chromophore peak, from the treatment with A. oryzae, was also smaller and slightly displaced; the same occurred with the other peaks. However, the absorbance values were higher in comparison to those of P. chrysosporium, indicating that no greater removal of the dye occurred after 240 h of treatment with A. oryzae. 3.1.2 Fourier Transform Infrared Analysis An analysis of the FTIR spectra is required to better appreciate the absorption ratio changes and breaking of molecule bonds by both fungi. The spectra measured correspond to 24 h (Fig. 3), 144 h (Fig. 5), and 240 h Fig. 2 Absorption spectra of dye solution after 240 h of interaction with P. chrysosporium (a) and A. oryzae (b) at 30±1 °C, with scans performed at 24, 144, and 240 h Water Air Soil Pollut (2014) 225:2026 Page 5 of 11, 2026 Fig. 3 FTIR spectrum of control dye solution and samples after 24 h of treatment with P. chrysosporium and A. oryzae (Fig. 6) following treatment with P. chrysosporium and A. oryzae. The most significant changes in the structure of the dye occurred in the 2,000 to 400 cm−1 range. Different bands from the control were found after the first 24 h of treatment, indicating changes in the structure of the dye following contact with the fungi. The most significant change regards the appearance of a new band at 1,116 cm−1 with A. oryzae. This region is characteristic of amine groups (Juárez-Hernández et al. 2008; Fanchiang and Tseng 2009). A. oryzae likely has enzymes capable of breaking any azo connection (N═N) within the first hours, enhancing the amine signal. Therefore, this band appeared and remained in the spectra at 144 and 240 h. The band in the 1,734 cm−1 range, which was not in the control, was found after treatment with both fungi. According to Wharfe et al. (2010) and Forato et al. (1998), the 1,700 to 1,500 cm−1 range corresponds to the protein, specifically the stretching of the C═O bond of the amide I peptide. This band likely appeared due to the presence of enzymes produced by P. chrysosporium, which has an important ligninolytic system, and those produced by A. oryzae, which is widely known for the synthesis of enzymes such as laccase. This band remained in all treatments analyzed. The band at 1,008 cm−1 was found in the control solution and the treatment with A. oryzae after 24 (Fig. 3), 144 (Fig. 5), and 240 h (Fig. 6). Stretching of the S═O bond of the sulfonic group occurs in this range (Silverstein et al. 1994; Alvares et al. 2006; Dhanve et al. 2009). As these bands were not evident in the spectrum with P. chrysosporium, it is believed that the enzymes of the A. oryzae were able to cause a change in the sulfonic group that P. chrysosporium could not. The band at 991 cm−1, which indicates C―O―H deformation (Robert et al. 2005), only occurred in the solutions treated with A. oryzae after 24, 144, and 240 h. The C―O bond is present in naphthol (Pham et al. 1997) and had a signal at 1,053 cm−1 in the samples. However, the signal intensified with P. chrysosporium, while the band performed with less intensity with A. oryzae. Therefore, the naphthol C―O―H bond was altered by both fungi, but to different extents. The bands at 881 and 869 cm−1 appeared less intense in the treated solutions than in the control solution, especially with A. oryzae. These bands are characteristic of deformation of the C―H bond of the aromatic ring (Barbosa 2007; Polunin et al. 2008; El-Kabbany et al. 2010). Thus, the action of A. oryzae on the dye was more effective in this structure after 24 h. 2026, Page 6 of 11 Water Air Soil Pollut (2014) 225:2026 Fig. 4 FTIR spectrum in 1,280 to 1,100 cm−1 range of control dye solution (a) and 24 h after treatment with P. chrysosporium (b) and A. oryzae (c) after Lorentzian deconvolution The same deformation of the C―H bond of aromatic ring occurred in the 582 cm−1 range of the control solution (Polunin et al. 2008). Again, the biggest change occurred in the spectrum with A. oryzae. The band at 594 cm−1 decreased in intensity, demonstrating a band at 617 cm−1 of the sulfonic acid group (Silverstein et al. 1994; Dhanve et al. 2009). The band in the 1,226 cm−1 range, corresponding to the stretching of the sulfonic group (Khaled et al. 2009) and the C═ S of thiocarbonyl group (Silverstein et al. 1994) was visible after both treatments, but apparently did not appear in the control. This was due to the overlapping of the bands at 1,225, 1,201, and 1,176 cm−1 in the control, visible only after deconvolution using the Lorentzian function (Forato et al. 1998) in the 1,280 to 1,100 cm−1 range. Figure 5 illustrates the deconvolution of the spectra of the control solution (Fig. 4a) and dye treated with P. chrysosporium (Fig. 4b) and A. oryzae (Fig. 4c). Lorentzian deconvolution allowed the identification of overlapping bands, demonstrating the band in the 1,225 cm−1 range in the control. The bands at 1,201 and 1,176 cm−1 correspond to the stretching of the C―N bond (Romão et al. 2003; Yadav et al. 2007; Barbosa 2007). However, only the band at 1,201 cm−1 remained constant after treatment with both fungi. The band at 1,176 cm−1 displayed lower intensity in the spectra of the dye after contact with P. chrysosporium and A. oryzae. This decrease in intensity was visible only with the deconvolution of the bands in the 1,280 to 1,150 cm−1 range. Water Air Soil Pollut (2014) 225:2026 Page 7 of 11, 2026 Fig. 5 FTIR spectrum of control dye solutions and samples after 144 h of treatment with P. chrysosporium and A. oryzae The lesser intensity at 1,173 cm−1 in the spectrum with P. chrysosporium (Fig. 4b) and displacement to 1,185 cm−1 in the spectrum with A. oryzae (Fig. 4c) indicate that the enzymes synthesized by the microorganisms acted on one or more C―N amine bonds, causing a change and even a possible breakage of these bonds. After 144 h of treatment (Fig. 5), early new bands in the 1,128, 1,109, and 1,037 cm−1 range occurred in the spectrum with P. chrysosporium. According to Fanchiang and Tseng (2009), these bands are characteristic of the C―NH2 bond. The appearance of primary amines in the treated solutions suggests that there was breakage of the azo bond. Another characteristic band of the deformation of C―N of the primary amine was found in the 833 cm−1 range, characteristic of the C―NH2 bond (Barbosa 2007; Telke et al. 2010). This low-intensity band appeared in the spectra with both fungi. As DB71 dye has three azo bonds, the appearance of signs of primary amines is expected to intensify, resulting from the breakage of these bonds during the biodegradation process. Another variation in the spectra occurred in the 1,400 cm−1 range. This band remained unchanged in relation to the control after 24 h (Fig. 3). After 144 h, however, intensification of the band occurred in the spectrum with A. oryzae (Fig. 5). This region is characterized by the C═C stretch of naphthalene derivatives (Barbosa 2007), the N═N stretch (Cervantes et al. 2009; Franciscon et al. 2009), and the presence of free amines (Wharfe et al. 2010). Thus, the action of enzymes from A. oryzae on the dye molecule interfered with the bonds and may cause their rupture, releasing amines into the solution. The other bands in the spectra remained apparently with the same intensity, except the band in the 597 cm−1 region, representing the deformation of the C―H bond of the aromatic ring (Polunin et al. 2008), which decreased in the spectrum with A. oryzae. Thus, the band at 619 cm−1 related to sulfonic groups (Dhanve et al. 2009; Silverstein et al. 1994) appeared more intensely than at 597 cm−1. The bands in the 1,008 and 991 cm−1 range, corresponding to the stretching of the S═O bond, were again found only in the spectrum with A. oryzae. Among the changes in the spectra 240 h after treatment (Fig. 6), the most evident was in the 1,400 cm−1 region with A. oryzae. A significant increase in the intensity of this band occurred. It is likely that the azo bonds were broken, as already mentioned, releasing primary amines. The dye is triazo and as this dye has an amine linked to a naphthol, the azo bonds of the 2026, Page 8 of 11 Water Air Soil Pollut (2014) 225:2026 Fig. 6 FTIR spectrum of control dye solution and samples after 240 h of treatment with P. chrysosporium and A. oryzae molecule broke and six new amines emerged; thus, the band appeared intensified. The band at 1,112 cm−1 was also enhanced, supporting the hypothesis of the breakage of bonds and demonstrating the degradation of the dye molecule. Observing the spectra, an absence of new bands was noted. However, the intensification of another band from the sulfonic group was observed at 619 cm−1 in the spectrum with A. oryzae. The dye has four sulfonate groups. It is possible that, after the breakage of some bonds, these groups may have intensified their signal, thereby increasing the intensity of characteristic bands. Evaluating the spectra of the treated solutions up to 240 h, A. oryzae caused the greatest changes in the spectra, indicating greater efficiency in the biodegradation process. This finding is due to the fact that the first 24 h of treatment were sufficient for the appearance of characteristic bands of amine, indicating the possible breakage of azo bonds. P. chrysosporium was also able to degrade the dye molecule, but required a longer time to achieve breakage. Telke et al. (2010) report that some species of Aspergillus are able to decolorize a wide range of structurally different dyes and are more effective than the widely studied basidiomycete P. chrysosporium. This is consistent with the findings of the present study for both biosorption and biodegradation. The analysis of all information collected by FTIR relating to the structure of the dye allows establishing a biodegradation pathway for the DB71 dye molecule. Figure 7 represents the molecule before biodegradation and the possible molecules formed after the process in an acid medium. Considering the major studies conducted on azo dyes, information on the biodegradation mechanism is limited (Telke et al. 2010). The FTIR analysis demonstrated that the biodegradation of DB71 dye does not occur equally with the two fungi tested. It is likely that these fungi synthesize a range of enzymes in certain quantities that interact differently. However, based on the molecules identified, the metabolites formed at the end of the biodegradation process suggest broken bonds (Fig. 7). Water Air Soil Pollut (2014) 225:2026 Page 9 of 11, 2026 Fig. 7 Pathway proposed for degradation of DB71 dye after treatment with P. chrysosporium and A. oryzae It should be stressed that the proposed pathway does not consider the complete mineralization of the dye. The mineralization may occur if other conditions for the biodegradation test are established, especially with regard to the time available for biodegradation to occur at higher speeds, thereby creating conditions for the breakdown of aromatic rings. However, there is little information on the mineralization of these compounds. Thus, the data generated in the present study are relevant to the advancement of studies on the biodegradation of dyes, especially azo dyes. 4 Conclusion UV-VIS spectrophotometer and FTIR analysis allowed the determination of the susceptibility of the DB71 dye to degradation by both fungi tested. Biodegradation was proven by the appearance of characteristic bands in the FTIR spectra of the expected metabolites stemming from the breakage of bonds in the dye. This process occurred unevenly between the two fungi. However, at the end of treatment (240 h), both A. oryzae and P. chrysosporium had degraded the dye molecules in solution generating similar metabolites. The treatment with A. oryzae demonstrated more significant signs of biodegradation in the first 24 h in comparison to P. chrysosporium. Thus, it is likely that enzymes from A. oryzae have greater affinity for the dye molecules. P. chrysosporium also proved effective, but not with the same potential as that of A. oryzae. Therefore, P. chrysosporium and A. oryzae demonstrate considerable potential for the treatment of textile effluents through biodegradation. Acknowledgments We thank CAPES (Coordination for Enhancement of Higher Education Personnel), CNPq (National Council for Scientific and Technological Development), and FAPESP (São Paulo Research Foundation) for financial support. 2026, Page 10 of 11 References Alvares, D.A., Moreira, J.C., Scuracchio, C.H., Onmori, R.K. (2006). Study of morphology and electrical properties of dEPDM rubber and PAni Blends. Proceedings of 17° Congresso Brasileiro de Engenharia e Ciência dos Materiais, Foz do Iguaçu, Brazil, 8330–8335. 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