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Decolorization of reactive azo dyes by Cunninghamella elegans UCP 542 under co-metabolic conditions
Bioresource Technology 91 (2004) 69–75
Decolorization of reactive azo dyes by Cunninghamella elegans UCP 542 under co-metabolic conditions S.T. Ambr osio a, G.M. Campos-Takaki a
a,b,*
Doutorado em Ci^ encias Biol ogicas, Universidade Federal de Pernambuco, 50670-420 Recife-Pernambuco, Brazil b Departamento de Quımica e N ucleo de Pesquisas em Ci^ encias Ambientais, Universidade Cat olica de Pernambuco, 50050-590 Recife-Pernambuco, Brazil Received 9 September 2002; received in revised form 15 May 2003; accepted 16 May 2003
Abstract The inappropriate disposal of dyes in wastewater constitutes an environmental problem and can cause damage to the ecosystem. Alternative treatments have been reported that fungi are particularly effective in the decolorization of textile effluents. The decolorization of dyes with different molecular structures by Cunninghamella elegans was evaluated under several media conditions. The decolorization procedures consisted of adding 72 h of mycelium into the culture medium containing either orange or reactive black or reactive red or a mixture of these dyes in the presence or absence of sucrose and/or peptone. The decolorization profile was highly dependent upon the incubation time, the molecular structure of the dye and presence or absence of co-substrates. The presence of sucrose or both sucrose and peptone significantly increased the decolorization of the solutions, however, the presence of only the nitrogen source suppressed it. The ultraviolet spectra of the solutions before and after decolorization suggested the occurrence of biodegradation in addition to the biosorption of the dyes. All tested dyes, except for the reactive black, caused inhibition of respiration of Escherichia coli, which suggested that toxic metabolites were produced. 2003 Elsevier Ltd. All rights reserved. Keywords: Zygomycetes; Biodegradation; Biosorption; Textile dyes; Toxicity
1. Introduction The reactive azo dyes are characterized by the presence of a nitrogen–nitrogen double bond (–N@N–), namely the azo group, which is bound to aromatic groups. Usually during the textile processing, around 30–70% of the amount of the dye used is hydrolyzed and eliminated into the wastewater (Bumpus, 1995). In order to meet the criteria necessary for industrial applications, these dyes present a diversity of colors, molecular structures and resistance to fading upon exposure to light, water and many chemical compounds (Correia et al., 1994). These required criteria thus yield compounds that cause serious environmental pollution problems. As a result of the environmental legislation, industries are being forced to treat dye contaminated their effluents (Robinson et al., 2001). Due to their genetic diversity and metabolic versatility, microorganisms * Corresponding author. Tel.: +55-81-32164017; fax: +55-8132714043. E-mail address: [email protected] (G.M. Campos-Takaki).
0960-8524/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-8524(03)00153-6
have become a viable alternative to remediate the pollution problem caused by reactive azo dyes (Alexander, 1994). Currently, bioremediation is becoming important because it is cost effective, environmentally friendly, and produces less sludge (Banat et al., 1996; Robinson et al., 2001). Many bacteria are able to degrade azo reactive dyes aerobically and anaerobically (Tan et al., 1999); however, in many cases the metabolic products, usually aromatic amines (Hu, 2001), are toxic or even more toxic than the starting azo dyes. Phanerochaete chrysosporium has been reported to efficiently degrade azo reactive dyes such as Orange II, Congo red e Tropaeolim (Cripps et al., 1990). In addition, it has recently been demonstrated that peroxidases (LiP and MnP), phenoloxidases (laccases), and dioxygenases can act on specific recalcitrant pollutants by precipitation or transforming them into other products, thus allowing for a possible better final treatment (Duran and Esposito, 2000). It has been shown that some species of Basidiomycetes such as Phlebia tremellosa (Kirby et al., 2000), Irpex lacteus, Pleurotus ostreaus (Novotny et al., 2001) and Trametes modesta (Nyanhongo et al., 2002), can
70
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
nambuco State, Brazil by Gomes et al. (2000). The isolation and identification of strain are according to OÕDonnell (1979). The culture was maintained on Sabouraud dextrose agar at 4 C and deposited in Culture Collection of Nucleus of Resource in Environmental Sciences, Catholic University of Pernambuco, Recife, PE, Brazil. Selected features of the azo dyes studied are presented in Table 1. The azo dyes C.I. reactive black-5 and C.I. reactive red-198 were provided by the Suape T^extil Co., located in the City of Cabo, Pernambuco, Brazil, and the orange II (C.I.15510) was obtained from Sigma (Sigma-Aldrich Corporation, St. Louis, Missouri, USA). Solutions of these dyes were prepared by dissolving a given amount of the powder in distilled water and then added to the medium in order to yield solutions with final concentrations of 0.025 mM for the pure dyes and 0.034 mM for the mixture of dyes.
play an important role in the azo dyes decolorization, despite the fact that the detailed biochemical pathways underlying the fungal degradation are not yet well understood (Banat et al., 1996; Slockar and Le Marechal, 1998; Zheng et al., 1999). In addition, it has also been demonstrated that fungi can utilize dye chemical substances such as aniline, as the sole source of carbon and nitrogen (Emtiazi et al., 2001). Consequently, the efficiency of the decolorization process can be improved by the addition of suitable co-substrates in the culture medium (Sumathi and Manju, 2000; Panswad and Luangdilok, 2000), significantly reducing the costs of the process (Kapdan et al., 2000). Studies on nonligninolytic fungi metabolizing dyes are minimal, and only recently, has there been a report that Cunninghamella elegans ATCC 36112 was able to metabolize 85% of the triphenylmethane dye malachite green after 24 h of incubation. The mechanism of this degradation process by C. elegans is yet to be elucidated; however, this fungus is capable of metabolizing a wide range of compounds, particularly by demethylation and oxidation (Cha et al., 2001). The main objective of the present study was to examine the decolorization of three reactive azo dyes and their mixture by mycelium of C. elegans in presence or absence of carbon and/or nitrogen sources, as well as the determination of the toxicity of dyes after the action of the fungus by reduction of Escherichia coli respiration.
2.2. Culture conditions The fungal cultures were grown in 250 ml Erlenmeyer flasks using spore suspensions (3.8 · 108 spores/ml) into 95 ml of Sabouraud broth medium sterilized at 121 C for 15 min (Hansen et al., 1995). For the decolorization experiments, 72 h old fungal mycelium was inoculated in modified Sabouraud broth media (Lacaz et al., 1991) in Table 2. Sucrose was used rather than glucose, as a more economical substitute (Kapdan et al., 2000). The pH of the medium was adjusted to 5.8 before sterilization and the culture was incubated in an orbital shaker at 150 rpm and 28 C. Aliquots were removed after 12, 24, 48, 72, 96, 120 and 168 h. Controls of the experiments were designed to perform under the same conditions described earlier without the fungi. All experiments were
2. Methods 2.1. Microorganism and dyes C. elegans UCP 542 was isolated from mangrove sediment collected in the City of Rio Formoso, Per-
Table 1 Structure of azo dyes and their respective wavelength maximum absorption (kmax ) Dyes
kmax (nm)
Orange II
485
Chemical structure OH NaO3S
Reactive black 5
N
N
597
OH NH2 NaO3 SOCH2 CH 2 O2 S
N N
N
N
NaO 3 S
Reactive red 198
SO3Na
517
H 3C NH N
SO3Na N
OH N
NH
N N Cl
NaO 3S
SO 3 Na
SO2 CH 2 CH2 OSO3 Na
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 Table 2 Composition of media used in the dye decolorization experiments Componentsa
Medium I
II
III
IV
Peptone (10 g/l) Sucrose (20 g/l) KH2 PO4 (0.5 g/l) Dye
X X X X
X – X X
– X X X
– – X X
a
71
structure, culture media, incubation period, and the interaction among them. The means of the significantly different main effects were compared by the DuncanÕs test at the 5% level using the Statistica program (Statsoft Inc., 1997).
3. Results and discussion
X denotes the presence of the component.
performed in triplicate and each treatment had four replicates. 2.3. Color reduction measurements Absorbance measurements were performed with a UV–VIS spectrophotometer (Spectronic Genesis, model 2-Spectronic Instruments Inc., USA). The wavelengths for the measurements were set at the values presented in Table 1 as kmax for the pure dyes, and at 511 nm for the mixture. Dye concentration was calculated from an absorbance x concentration calibration curve. All calibration curves yielded a linear range 0.005–0.025 mM/l and a linear regression coefficient at least 0.99. 2.4. Assessment of acute toxicity The toxicity tests were performed at the Biological Chemistry Laboratory of University of Campinas (S~ao Paulo, Brazil). The indicator organism, E. coli ATCC 25922, was provided by Culture Collections of Tropical Fundation Andre Tosello (University of Campinas, S~ao Paulo, Brazil). The bioassay was based on the inhibition of respiration of the bacteria by pollutant and was carried out on a clear supernatant after fungal treatment in comparison to the toxicity of controls. The assay involves the incubation of E. coli cultures at 37 C with known amounts of the pollutant (solution-dyes). When the CO2 concentration produced by microbial respiration reached 0.5 mmol/l, 45 ml of the cultures of the E. coli was transferred into several flasks and each one received 5 ml of one sample withdrawn at a selected time. As a control, 5 ml of distilled water was introduced in one the flasks and the CO2 production monitored every 20 min using flow injection analysis (FIA). The test is described in detail in Moraes et al. (2000). 2.5. Statistical analysis To evaluate the influence of the media components upon the decolorization of dye solutions the ANOVA, analysis of variance of the data, was performed, which involved the pre-treatment of the data by the arcsine function, namely, arcsine (x=100), with x being the original data. The main factors analyzed were the dye
The azo reactive dyes black-5, red-198, orange II were chosen for this study since they are widely used in the cotton textile industry in Brazil and throughout the world. Four different media have been used in order to establish the most suitable conditions for decolorization of solutions containing dyes and their mixture by the mycelium of C. elegans. This fungus was capable of decolorizing all dyes and the respective combinations in all media were studied. The ANOVA analysis of the data indicated that the decolorizations were statistically significant (P < 0:05) for all media and dyes (Fig. 1). For simplicity, representative values presented data pooled, and the distinction between the averages was indicated by combination of different letters. These results showed that for this particular fungus the decolorization of the dyes solutions could be selectively supported by the media composition. For instance, the medias I and III were more appropriate for decolorization of the orange II dye, whereas medium II and IV were more suitable for decolorization of reactive red and mixture of dyes. In addition, the mixture decolorization was strongly dependent upon the medium respectively used in the procedure. The decolorization produced by the fungus was highly selective despite the fact that all dyes process the same azo-stilbene moiety. A relationship between the selectivity induced by the medium and the molecular structure of the dyes might be established if it is consider that medias I and III contain carbon sources leading to a significant (P < 0:05) increase of the biomass (50% and 70%, respectively). Consequently the metabolism and the biosorption of the orange II dye should be preferred, since it is the poorest on carbon source, as well as the smallest molecule, its small steric hindrance should allow for a better biosorption. It should however be noted that the relationship between the molecular structure of the dyes and their decolorization by fungal treatment is still not clearly established (Knapp et al., 1995). Quantitatively, it was observed that in the presence of sucrose and peptone (medium I), the decolorization of orange II was nearly 83% after 96 h, whereas the decolorization of the other dyes not as significant (Fig. 1A). Conversely, in the presence of peptone as the sole source of organic nitrogen (medium II) the decolorization was not satisfactory, since the most significant decolorization (48% after 72 h) was achieved for the
72
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 Medium II
0.65 a
1.0
Medium I
0.9
0.55
0.8
0.50
a
0.6
b
b
0.5 b a
0.3
ab a
b ab bb
b
bcc
a
a
b
a
0.35 ab
0.30
bc
0.25
c
c ab b
b
0.05
c
d
d
a b
b
c
ab
0.20 ab
b c
c
a
0.10
0.0
b
b
0.40
0.15
a
ab b
0.1
a
b
a
a
b
ab
ab
0.4
a
b b
Decolorization (%)
Decolorization (%)
a
0.45
0.7
0.2
a
0.60
0.00 12
24
72
48
(A)
96
12
168
120
24
72
48
(B)
Time (hours)
96
Time (hours)
Medium III 1.4
Medium IV
a
a
a
168
120
1.0
a
a
1.2
Decolorization (%)
Decolorization (%)
0.8 a
a
1.0 0.8 a
b
ab
0.6
b
b 0.4
ab
b
b
b b
b
bb b
b
a
a 0.4
bb
c
c
a
b
a
b ab bc
bb
0.0
b
c
c b
d
c
b
0.0 12
(C)
a
a
b
a
a
0.2 aa
a c
0.6
bc
b
a 0.2
a
24
48
72
96
120
168
Time (hours)
12
(D)
24
48
72
96
120
168
Time (hours)
Fig. 1. Effect of medium composition I decolorization of the dyes orange II (j), reactive black (), reactive red ( ) and mixture ( ) by C. elegans 542. The letters indicate average values which are statistically different at P ¼ 0:05, using the DuncanÕ test.
reactive red (Fig. 1B). These results showed that the presence of peptone interfered in the color removal of the azo dye, as has already been reported (Tatarko and Bumpus, 1998; Zheng et al., 1999; Fu and Viraraghavan, 2001) concerning the color removal reduction due to the presence of inorganic nitrogen, such as ammonium ions. Satisfactory decolorization was obtained for all dyes in the medium III, which contained only sucrose (Fig. 1C). Comparisons with medium I, which contained sucrose and peptone, exhibited distinct behaviors. The decolorization of orange II was slightly greater as well as faster for medium III (88% after 96 h of incubation), since it was significant just after 48 h of incubation. These differences can be interpreted as a negative in-
terference of peptone (medium I), as observed in medium II (peptone only). For comparison, a medium without sucrose and peptone (medium IV) was used and a distinct behavior for the decolorization was observed (Fig. 1D). In this medium, the decolorization of orange II (10%), reactive black (40%) and mixture of dyes (40%) was slight even after 72 h of incubation when compared with media containing sucrose. However, a significant decolorization for the reactive red dye (80%) was achieved after 120 h of incubation. These results thus showed that the medium composition was important for the decolorization of dyes by this fungus treatment and that selective decolorization can be obtained by combining different conditions.
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
70
60
Toxicity (%)
50
40
30
20
10
0 Orange II
Reactive black
Reactive red
Mixture
Dyes
Fig. 2. Toxicity of dyes to E. coli before (j) and after () treatment with C. elegans UCP 542.
structures of the crystal violet and reactive black are very different as should be their metabolites. The UV–VIS spectra results, are presented in Fig. 3. The normalized spectra of the orange II dye solutions before (control) and after the fungal treatment under all media conditions were studied. It is apparent that for medias I and II, in which the descolorization of the orange II solutions were significant, the UV–VIS spectra exhibit a shift of the maximum of absorption towards shorter wavelengths upon fungal treatment. This indicates that decolorization of this dye solution occurred by degradation in addition to the visual observation of the biosorption process (Wang and Yu, 1998). For the decolorization in medias II and IV, the spectral shifts in
Medium II Medium I Medium III
1.0
Medium IV
0.8 Control Absorbance
An additional observation should also be reported regarding the physical appearance of the samples. Namely, the samples on medium containing sucrose exhibited biomass production with a strong orange color after 24–48 h of incubation; this strong color became faint after 96 h and then returned to the original appearance of the mycelium prior to treatment. However, the color of the solution remained unchanged during this observation, what suggests that the dye molecules adsorbed on the biomass were not released to the solution (desorption), being probably degraded during the fungal metabolism. Comparisons with results reported in the literature (Fu and Viraraghavan, 2001) corroborate most of the observations for the decolorization of the reactive azo-dye by fungal treatment. As mentioned previously, the chemical structures of the dye molecules are important in decolorization (Novotn y et al., 2001) and no clear relationships have been established between the molecular structure, the amount and the selectivity of the decolorization. For instance, Aspergillus foetidus was unable to utilize drimared red and drimared blue dyes as the sole source of energy, whereas in the presence of sucrose a color removal of 95–99% was obtained (Sumathi and Manju, 2000). The dye everzol turquoise blue was 98% decolorized by Coriolus versicolor in presence of sucrose and urea (Kapdan et al., 2000). Despite the observations mentioned before, it has not been clearly established if the present fungal treatment involved the degradation of the dyes in addition to the visually observed biosorption. Thus, two techniques were used to provide some indications of the presence or absence of degradation, namely, toxicity tests and UV– VIS spectra. Toxicity tests showed that the solutions resulting from the fungal treatment of all tested azo dyes, except for the reactive black, inhibited the respiration of E. Coli when compared to the untreated solutions. This was an indication that the fungus was producing metabolites and thus the degradation was active (Fig. 2). It is known that several enzymes are present in the microsomal and cytosolic fractions of C. elegans (Wackett and Gibson, 1982; Zhang et al., 1996). Most of these enzymes are oxidative mechanism, particularly the cytochrome P-450, which might use the dye molecules as substrate rendering them colorless after the reaction. It has already been reported that the biodegradation of crystal violet by C. elegans did not produce metabolites as toxic as the starting dye (Cha et al., 2001), whereas the reductive azo linkage by bacteria resulted in the formation of aromatic amines, which can be highly toxic and carcinogenic (Hu, 2001). Our results have shown that the solution resulting from the fungal treatment of the medium containing the reactive black did not inhibit the respiration of E. coli. This analogy should not be taken any further since the molecular
73
0.6
0.4
0.2
0.0 300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 3. UV–VIS spectra of orange II azo dye before and after the treatment with C. elegans UCP 542 on different media: control ( ), medium I ( ), medium II (––), medium III ( ) and medium IV ( ).
74
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
the UV–VIS region were not significant and thus the dominant mechanism for decolorization would be the biosorption of the dye molecules (Zheng et al., 1999). Regarding the spectral shifts towards shorter wavelengths observed in the treatment with medias I and III, they were probably produced by the biodegradation of the dye molecule that led to a decrease of the conjugation effects between the aromatic rings. It should be noted that for conjugated systems the wavelength of the maximum absorption was very sensitive to the size of the conjugation, where a decrease of one unit of double bond can lead to shifts in range of 25–30 nm in polyeniccarbonyl conjugated systems (Streitwieser et al., 1992). In Fig. 4 we presented the UV–VIS spectra of the solution containing a mixture of all dyes before and after the fungal treatment. It can be readily observed that the decolorization that had already occurred just after 24 h is mainly due to biosorption, since there were no spectral differences between the control and the solution after the treatment. However, the solution after 96 h became re-colorized, due to some products from desorption, but mainly due to the degradation of the adsorbed dyes, since there were significant spectral shifts in the 500 and 400 nm regions. Thus, the results of the UV–VIS spectra corroborated the toxicity tests regarding the degradation of the dyes by C. elegans in the presence of sucrose. In addition, these data showed that it was viable to use UV–VIS spectroscopy to determine the kinetics of the biosorption and the degradation processes during the decolorization. In conclusion, the decolorization process produced by treatment with C. elegans was strongly dependent upon the co-metabolism conditions, where the sucrose enhanced and peptone had a negative effect upon the decolorization. This decolorization process was also dependent upon the molecular structure of the Mixture 0.6
Absorbance
0.5 0.4 Control 0.3 Mixture after 24 h 0.2
Mixture after 96 h
0.1 0.0 300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 4. UV–VIS spectra of the mixture of dyes, before ( ) and after 24 (- - -) and 96 ( ) h of treatment with C. elegans UCP 542 on condition medium III.
dye, despite the fact that the studied dyes had a common stilbene-azo structural motif. In addition, it has been shown by toxicity tests and UV–VIS spectral analysis that the decolorization process involved biodegradation, in addition to the visually observed biosorption.
Acknowledgements The authors gratefully acknowledge Prof. Dr. Ricardo L. Longo (DQF/UFPE) for assistance on analytical interpretation of the experimental data and review of the manuscript; Dra. Sandra G. Moraes (IQM/UNICAMP, SP) for the toxicity tests and Dra. Adriana M.Y. Melo (Pesquisadora CNPq/Embrapa rido) for helping with the statistical analyses. We Semi-A would like to acknowledge the Brazilian agencies CNPq, CAPES, the programs FINEP/CTPETRO/AVINA GROUP and PRONEX for financial support. References Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press. p. 302. Banat, I.M., Nigam, P., Singh, D., Marchant, R., 1996. Microbial decolorization of textile-dye containing effluents: a review. Bioresource Technology 58, 217–227. Bumpus, J.A., 1995. Microbial degradation of azo dyes. In: Singh, V.P. (Ed.), Biotransformations: Microbial degradation of healthrisk compounds. Elsevier Science, Amsterdam, pp. 157–175. Cha, C.J., Doerge, D.R., Cerniglia, C.E., 2001. Biotransformation of malachite green by fungus Cunninghamella elegans. Applied and Environmental Microbiology 67 (9), 4358–4360. Cripps, C., Bumpus, J.A., Aust, S.D., 1990. Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Applied Environmental Microbiology 56 (4), 1114–1118. Correia, V.M., Stephenson, T., Judd, S., 1994. Characterisation of textile wastewaters––a review. Environmental Technology 15, 917– 929. Duran, N., Esposito, E., 2000. Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Applied Catalysis B: Environmental 28, 83–99. Emtiazi, G., Satarii, M., Mazaherion, F., 2001. The utilization of aniline, chlorinated aniline, and aniline blue as the only source of nitrogen by fungi in water. Water Research 35 (5), 1219–1224. Fu, Y., Viraraghavan, T., 2001. Fungal decolorization of dye wastewaters: a review. Bioresource Technology 79, 251–262. Gomes, P.F., Nascimento, A.E., Okada, K., Messias, A.S., Sharia, A.E.N., Campos-Takaki, G.M., 2000. Aspectos da qualidade de ecossistemas do Municıpio do Rio Formoso. International Conference––Sustainable use of estuaries and mangroves: challenges and prospects, p. 82. Hansen, E.B., Cho, B.P., Korfmacher, W.A., Cerniglia, C.E., 1995. Fungal transformation of antihistamines: metabolism of brompheniramine, chlorpheniramine, and pheniramine to N-oxide and Ndemethylated metabolites by fungus Cunninghamella elegans. Xenobiotica 25 (10), 1081–1092. Hu, T.L., 2001. Kinetics of azoreductase and assessment of toxicity of metabolic products from azo dye by Pseudomonas luteola. Water Science and Technology 43 (2), 261–269. Kapdan, I.K., Kargi, F., McMullan, G., Marchant, R., 2000. Effect of environmental conditions on biological decolorization of textile
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 dye by C. versicolor. Enzyme and Microbial Technology 26, 381– 387. Kirby, N., Marchant, R., McMullan, G., 2000. Decolourisation of synthetic textile dyes by Phlebia tremellosa. FEMS Microbiology Letters 188, 93–96. Knapp, J.S., Newby, P.S., Reece, L.P., 1995. Decolorization of dyes by wood-rotting basidiomycete fungi. Enzyme and Microbial Technology 17, 664–668. Lacaz, C.S., Porto, E., Martins, J.E., 1991. Micologia Medica: fungos, actinomicetos e algas de interesse medico. 8th. S~ao Paulo: Savier, p. 695. Moraes, S.G., Freire, R.S., Duran, N., 2000. Degradation and toxicity reduction of textile effluent by combined photocatalytic and ozonation processes. Chemosphere 40, 369–373. asek, V., Molitoris, Novotn y, C., Rawal, B., Bhatt, M., Patel, M., S H.P., 2001. Capacity of Irpex lacteus and Pleurotus ostreaus for decolorization of chemically different dyes. Journal of Biotechnology 89, 113–122. Nyanhongo, G.S., Gomes, J., G€ uubitz, G.M., Zvauya, R., Read, J., Steiner, W., 2002. Decolorization of textile dyes by lacases from a newly isolated strain of Trametes modesta. Water Research 36, 1449–1456. OÕDonnell, K.L., 1979. Zygomycetes in Culture. Palfrey Contributions in Botany, no. 2. Department of Botany, University of Georgia, Athens, p. 257. Panswad, T., Luangdilok, W., 2000. Decolorization of reactive dyes with different molecular structures under different environmental conditions. Water Research 34 (17), 4177–4184. Robinson, T., McMullan, G., Marchant, R., Nigam, P., 2001. Remediation of dyes in textile effluent: a critical review on current
75
treatment technologies with a proposed alternative. Bioresource Technology 77, 247–255. Slockar, Y.M., Le Marechal, A.M., 1998. Methods of decoloration of textile wastewaters. Dye and Pigments 37 (4), 335–356. Statsoft Inc––Statistica for Windows. Computer Program Manual. Tulsa, 1997. Streitwieser, A.C.H., Heathcock, E., Kosower, M., 1992. Introduction to Organic Chemistry, fourth ed. Macmillan Publishing Co., New York. p. 673. Sumathi, S., Manju, B.S., 2000. Uptake of reactive textile dyes by Aspergillus foetidus. Enzyme and Microbial Technology 27, 347– 355. Tan, N.C.G., Lettinga, G.H., Field, J.A., 1999. Reduction of the azo dye mordant orange 1 by methanogenic granular sludge exposed to oxygen. Bioresource Technology 67, 35–42. Tatarko, M., Bumpus, J.A., 1998. Biodegradation of congo red by Phanerochaete chrysosporium. Water Research 32 (5), 1713–1717. Wackett, L.P., Gibson, D., 1982. Metabolism of xenobiot compounds by enzymes in cell extracts of the fungus Cunninghamella elegans. Biochemical Journal 205, 117–122. Wang, Y., Yu, J., 1998. Adsorption and degradation of synthetic dyes on the mycelium of Trametes versicolor. Water Science Technology 38 (4–5), 233–238. Zhang, D., Yang, Y., Leakey, J.E.A., Cerniglia, C.E., 1996. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiology Letters 138, 221– 226. Zheng, Z.X., Levin, R.E., Pinkham, J.L., Shetty, K., 1999. Decolorization of polymeric dyes by a novel Penicillium isolate. Process Biochemistry 34 (1), 31–37.
Decolorization of reactive azo dyes by Cunninghamella elegans UCP 542 under co-metabolic conditions S.T. Ambr osio a, G.M. Campos-Takaki a
a,b,*
Doutorado em Ci^ encias Biol ogicas, Universidade Federal de Pernambuco, 50670-420 Recife-Pernambuco, Brazil b Departamento de Quımica e N ucleo de Pesquisas em Ci^ encias Ambientais, Universidade Cat olica de Pernambuco, 50050-590 Recife-Pernambuco, Brazil Received 9 September 2002; received in revised form 15 May 2003; accepted 16 May 2003
Abstract The inappropriate disposal of dyes in wastewater constitutes an environmental problem and can cause damage to the ecosystem. Alternative treatments have been reported that fungi are particularly effective in the decolorization of textile effluents. The decolorization of dyes with different molecular structures by Cunninghamella elegans was evaluated under several media conditions. The decolorization procedures consisted of adding 72 h of mycelium into the culture medium containing either orange or reactive black or reactive red or a mixture of these dyes in the presence or absence of sucrose and/or peptone. The decolorization profile was highly dependent upon the incubation time, the molecular structure of the dye and presence or absence of co-substrates. The presence of sucrose or both sucrose and peptone significantly increased the decolorization of the solutions, however, the presence of only the nitrogen source suppressed it. The ultraviolet spectra of the solutions before and after decolorization suggested the occurrence of biodegradation in addition to the biosorption of the dyes. All tested dyes, except for the reactive black, caused inhibition of respiration of Escherichia coli, which suggested that toxic metabolites were produced. 2003 Elsevier Ltd. All rights reserved. Keywords: Zygomycetes; Biodegradation; Biosorption; Textile dyes; Toxicity
1. Introduction The reactive azo dyes are characterized by the presence of a nitrogen–nitrogen double bond (–N@N–), namely the azo group, which is bound to aromatic groups. Usually during the textile processing, around 30–70% of the amount of the dye used is hydrolyzed and eliminated into the wastewater (Bumpus, 1995). In order to meet the criteria necessary for industrial applications, these dyes present a diversity of colors, molecular structures and resistance to fading upon exposure to light, water and many chemical compounds (Correia et al., 1994). These required criteria thus yield compounds that cause serious environmental pollution problems. As a result of the environmental legislation, industries are being forced to treat dye contaminated their effluents (Robinson et al., 2001). Due to their genetic diversity and metabolic versatility, microorganisms * Corresponding author. Tel.: +55-81-32164017; fax: +55-8132714043. E-mail address: [email protected] (G.M. Campos-Takaki).
0960-8524/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-8524(03)00153-6
have become a viable alternative to remediate the pollution problem caused by reactive azo dyes (Alexander, 1994). Currently, bioremediation is becoming important because it is cost effective, environmentally friendly, and produces less sludge (Banat et al., 1996; Robinson et al., 2001). Many bacteria are able to degrade azo reactive dyes aerobically and anaerobically (Tan et al., 1999); however, in many cases the metabolic products, usually aromatic amines (Hu, 2001), are toxic or even more toxic than the starting azo dyes. Phanerochaete chrysosporium has been reported to efficiently degrade azo reactive dyes such as Orange II, Congo red e Tropaeolim (Cripps et al., 1990). In addition, it has recently been demonstrated that peroxidases (LiP and MnP), phenoloxidases (laccases), and dioxygenases can act on specific recalcitrant pollutants by precipitation or transforming them into other products, thus allowing for a possible better final treatment (Duran and Esposito, 2000). It has been shown that some species of Basidiomycetes such as Phlebia tremellosa (Kirby et al., 2000), Irpex lacteus, Pleurotus ostreaus (Novotny et al., 2001) and Trametes modesta (Nyanhongo et al., 2002), can
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S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
nambuco State, Brazil by Gomes et al. (2000). The isolation and identification of strain are according to OÕDonnell (1979). The culture was maintained on Sabouraud dextrose agar at 4 C and deposited in Culture Collection of Nucleus of Resource in Environmental Sciences, Catholic University of Pernambuco, Recife, PE, Brazil. Selected features of the azo dyes studied are presented in Table 1. The azo dyes C.I. reactive black-5 and C.I. reactive red-198 were provided by the Suape T^extil Co., located in the City of Cabo, Pernambuco, Brazil, and the orange II (C.I.15510) was obtained from Sigma (Sigma-Aldrich Corporation, St. Louis, Missouri, USA). Solutions of these dyes were prepared by dissolving a given amount of the powder in distilled water and then added to the medium in order to yield solutions with final concentrations of 0.025 mM for the pure dyes and 0.034 mM for the mixture of dyes.
play an important role in the azo dyes decolorization, despite the fact that the detailed biochemical pathways underlying the fungal degradation are not yet well understood (Banat et al., 1996; Slockar and Le Marechal, 1998; Zheng et al., 1999). In addition, it has also been demonstrated that fungi can utilize dye chemical substances such as aniline, as the sole source of carbon and nitrogen (Emtiazi et al., 2001). Consequently, the efficiency of the decolorization process can be improved by the addition of suitable co-substrates in the culture medium (Sumathi and Manju, 2000; Panswad and Luangdilok, 2000), significantly reducing the costs of the process (Kapdan et al., 2000). Studies on nonligninolytic fungi metabolizing dyes are minimal, and only recently, has there been a report that Cunninghamella elegans ATCC 36112 was able to metabolize 85% of the triphenylmethane dye malachite green after 24 h of incubation. The mechanism of this degradation process by C. elegans is yet to be elucidated; however, this fungus is capable of metabolizing a wide range of compounds, particularly by demethylation and oxidation (Cha et al., 2001). The main objective of the present study was to examine the decolorization of three reactive azo dyes and their mixture by mycelium of C. elegans in presence or absence of carbon and/or nitrogen sources, as well as the determination of the toxicity of dyes after the action of the fungus by reduction of Escherichia coli respiration.
2.2. Culture conditions The fungal cultures were grown in 250 ml Erlenmeyer flasks using spore suspensions (3.8 · 108 spores/ml) into 95 ml of Sabouraud broth medium sterilized at 121 C for 15 min (Hansen et al., 1995). For the decolorization experiments, 72 h old fungal mycelium was inoculated in modified Sabouraud broth media (Lacaz et al., 1991) in Table 2. Sucrose was used rather than glucose, as a more economical substitute (Kapdan et al., 2000). The pH of the medium was adjusted to 5.8 before sterilization and the culture was incubated in an orbital shaker at 150 rpm and 28 C. Aliquots were removed after 12, 24, 48, 72, 96, 120 and 168 h. Controls of the experiments were designed to perform under the same conditions described earlier without the fungi. All experiments were
2. Methods 2.1. Microorganism and dyes C. elegans UCP 542 was isolated from mangrove sediment collected in the City of Rio Formoso, Per-
Table 1 Structure of azo dyes and their respective wavelength maximum absorption (kmax ) Dyes
kmax (nm)
Orange II
485
Chemical structure OH NaO3S
Reactive black 5
N
N
597
OH NH2 NaO3 SOCH2 CH 2 O2 S
N N
N
N
NaO 3 S
Reactive red 198
SO3Na
517
H 3C NH N
SO3Na N
OH N
NH
N N Cl
NaO 3S
SO 3 Na
SO2 CH 2 CH2 OSO3 Na
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 Table 2 Composition of media used in the dye decolorization experiments Componentsa
Medium I
II
III
IV
Peptone (10 g/l) Sucrose (20 g/l) KH2 PO4 (0.5 g/l) Dye
X X X X
X – X X
– X X X
– – X X
a
71
structure, culture media, incubation period, and the interaction among them. The means of the significantly different main effects were compared by the DuncanÕs test at the 5% level using the Statistica program (Statsoft Inc., 1997).
3. Results and discussion
X denotes the presence of the component.
performed in triplicate and each treatment had four replicates. 2.3. Color reduction measurements Absorbance measurements were performed with a UV–VIS spectrophotometer (Spectronic Genesis, model 2-Spectronic Instruments Inc., USA). The wavelengths for the measurements were set at the values presented in Table 1 as kmax for the pure dyes, and at 511 nm for the mixture. Dye concentration was calculated from an absorbance x concentration calibration curve. All calibration curves yielded a linear range 0.005–0.025 mM/l and a linear regression coefficient at least 0.99. 2.4. Assessment of acute toxicity The toxicity tests were performed at the Biological Chemistry Laboratory of University of Campinas (S~ao Paulo, Brazil). The indicator organism, E. coli ATCC 25922, was provided by Culture Collections of Tropical Fundation Andre Tosello (University of Campinas, S~ao Paulo, Brazil). The bioassay was based on the inhibition of respiration of the bacteria by pollutant and was carried out on a clear supernatant after fungal treatment in comparison to the toxicity of controls. The assay involves the incubation of E. coli cultures at 37 C with known amounts of the pollutant (solution-dyes). When the CO2 concentration produced by microbial respiration reached 0.5 mmol/l, 45 ml of the cultures of the E. coli was transferred into several flasks and each one received 5 ml of one sample withdrawn at a selected time. As a control, 5 ml of distilled water was introduced in one the flasks and the CO2 production monitored every 20 min using flow injection analysis (FIA). The test is described in detail in Moraes et al. (2000). 2.5. Statistical analysis To evaluate the influence of the media components upon the decolorization of dye solutions the ANOVA, analysis of variance of the data, was performed, which involved the pre-treatment of the data by the arcsine function, namely, arcsine (x=100), with x being the original data. The main factors analyzed were the dye
The azo reactive dyes black-5, red-198, orange II were chosen for this study since they are widely used in the cotton textile industry in Brazil and throughout the world. Four different media have been used in order to establish the most suitable conditions for decolorization of solutions containing dyes and their mixture by the mycelium of C. elegans. This fungus was capable of decolorizing all dyes and the respective combinations in all media were studied. The ANOVA analysis of the data indicated that the decolorizations were statistically significant (P < 0:05) for all media and dyes (Fig. 1). For simplicity, representative values presented data pooled, and the distinction between the averages was indicated by combination of different letters. These results showed that for this particular fungus the decolorization of the dyes solutions could be selectively supported by the media composition. For instance, the medias I and III were more appropriate for decolorization of the orange II dye, whereas medium II and IV were more suitable for decolorization of reactive red and mixture of dyes. In addition, the mixture decolorization was strongly dependent upon the medium respectively used in the procedure. The decolorization produced by the fungus was highly selective despite the fact that all dyes process the same azo-stilbene moiety. A relationship between the selectivity induced by the medium and the molecular structure of the dyes might be established if it is consider that medias I and III contain carbon sources leading to a significant (P < 0:05) increase of the biomass (50% and 70%, respectively). Consequently the metabolism and the biosorption of the orange II dye should be preferred, since it is the poorest on carbon source, as well as the smallest molecule, its small steric hindrance should allow for a better biosorption. It should however be noted that the relationship between the molecular structure of the dyes and their decolorization by fungal treatment is still not clearly established (Knapp et al., 1995). Quantitatively, it was observed that in the presence of sucrose and peptone (medium I), the decolorization of orange II was nearly 83% after 96 h, whereas the decolorization of the other dyes not as significant (Fig. 1A). Conversely, in the presence of peptone as the sole source of organic nitrogen (medium II) the decolorization was not satisfactory, since the most significant decolorization (48% after 72 h) was achieved for the
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S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 Medium II
0.65 a
1.0
Medium I
0.9
0.55
0.8
0.50
a
0.6
b
b
0.5 b a
0.3
ab a
b ab bb
b
bcc
a
a
b
a
0.35 ab
0.30
bc
0.25
c
c ab b
b
0.05
c
d
d
a b
b
c
ab
0.20 ab
b c
c
a
0.10
0.0
b
b
0.40
0.15
a
ab b
0.1
a
b
a
a
b
ab
ab
0.4
a
b b
Decolorization (%)
Decolorization (%)
a
0.45
0.7
0.2
a
0.60
0.00 12
24
72
48
(A)
96
12
168
120
24
72
48
(B)
Time (hours)
96
Time (hours)
Medium III 1.4
Medium IV
a
a
a
168
120
1.0
a
a
1.2
Decolorization (%)
Decolorization (%)
0.8 a
a
1.0 0.8 a
b
ab
0.6
b
b 0.4
ab
b
b
b b
b
bb b
b
a
a 0.4
bb
c
c
a
b
a
b ab bc
bb
0.0
b
c
c b
d
c
b
0.0 12
(C)
a
a
b
a
a
0.2 aa
a c
0.6
bc
b
a 0.2
a
24
48
72
96
120
168
Time (hours)
12
(D)
24
48
72
96
120
168
Time (hours)
Fig. 1. Effect of medium composition I decolorization of the dyes orange II (j), reactive black (), reactive red ( ) and mixture ( ) by C. elegans 542. The letters indicate average values which are statistically different at P ¼ 0:05, using the DuncanÕ test.
reactive red (Fig. 1B). These results showed that the presence of peptone interfered in the color removal of the azo dye, as has already been reported (Tatarko and Bumpus, 1998; Zheng et al., 1999; Fu and Viraraghavan, 2001) concerning the color removal reduction due to the presence of inorganic nitrogen, such as ammonium ions. Satisfactory decolorization was obtained for all dyes in the medium III, which contained only sucrose (Fig. 1C). Comparisons with medium I, which contained sucrose and peptone, exhibited distinct behaviors. The decolorization of orange II was slightly greater as well as faster for medium III (88% after 96 h of incubation), since it was significant just after 48 h of incubation. These differences can be interpreted as a negative in-
terference of peptone (medium I), as observed in medium II (peptone only). For comparison, a medium without sucrose and peptone (medium IV) was used and a distinct behavior for the decolorization was observed (Fig. 1D). In this medium, the decolorization of orange II (10%), reactive black (40%) and mixture of dyes (40%) was slight even after 72 h of incubation when compared with media containing sucrose. However, a significant decolorization for the reactive red dye (80%) was achieved after 120 h of incubation. These results thus showed that the medium composition was important for the decolorization of dyes by this fungus treatment and that selective decolorization can be obtained by combining different conditions.
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
70
60
Toxicity (%)
50
40
30
20
10
0 Orange II
Reactive black
Reactive red
Mixture
Dyes
Fig. 2. Toxicity of dyes to E. coli before (j) and after () treatment with C. elegans UCP 542.
structures of the crystal violet and reactive black are very different as should be their metabolites. The UV–VIS spectra results, are presented in Fig. 3. The normalized spectra of the orange II dye solutions before (control) and after the fungal treatment under all media conditions were studied. It is apparent that for medias I and II, in which the descolorization of the orange II solutions were significant, the UV–VIS spectra exhibit a shift of the maximum of absorption towards shorter wavelengths upon fungal treatment. This indicates that decolorization of this dye solution occurred by degradation in addition to the visual observation of the biosorption process (Wang and Yu, 1998). For the decolorization in medias II and IV, the spectral shifts in
Medium II Medium I Medium III
1.0
Medium IV
0.8 Control Absorbance
An additional observation should also be reported regarding the physical appearance of the samples. Namely, the samples on medium containing sucrose exhibited biomass production with a strong orange color after 24–48 h of incubation; this strong color became faint after 96 h and then returned to the original appearance of the mycelium prior to treatment. However, the color of the solution remained unchanged during this observation, what suggests that the dye molecules adsorbed on the biomass were not released to the solution (desorption), being probably degraded during the fungal metabolism. Comparisons with results reported in the literature (Fu and Viraraghavan, 2001) corroborate most of the observations for the decolorization of the reactive azo-dye by fungal treatment. As mentioned previously, the chemical structures of the dye molecules are important in decolorization (Novotn y et al., 2001) and no clear relationships have been established between the molecular structure, the amount and the selectivity of the decolorization. For instance, Aspergillus foetidus was unable to utilize drimared red and drimared blue dyes as the sole source of energy, whereas in the presence of sucrose a color removal of 95–99% was obtained (Sumathi and Manju, 2000). The dye everzol turquoise blue was 98% decolorized by Coriolus versicolor in presence of sucrose and urea (Kapdan et al., 2000). Despite the observations mentioned before, it has not been clearly established if the present fungal treatment involved the degradation of the dyes in addition to the visually observed biosorption. Thus, two techniques were used to provide some indications of the presence or absence of degradation, namely, toxicity tests and UV– VIS spectra. Toxicity tests showed that the solutions resulting from the fungal treatment of all tested azo dyes, except for the reactive black, inhibited the respiration of E. Coli when compared to the untreated solutions. This was an indication that the fungus was producing metabolites and thus the degradation was active (Fig. 2). It is known that several enzymes are present in the microsomal and cytosolic fractions of C. elegans (Wackett and Gibson, 1982; Zhang et al., 1996). Most of these enzymes are oxidative mechanism, particularly the cytochrome P-450, which might use the dye molecules as substrate rendering them colorless after the reaction. It has already been reported that the biodegradation of crystal violet by C. elegans did not produce metabolites as toxic as the starting dye (Cha et al., 2001), whereas the reductive azo linkage by bacteria resulted in the formation of aromatic amines, which can be highly toxic and carcinogenic (Hu, 2001). Our results have shown that the solution resulting from the fungal treatment of the medium containing the reactive black did not inhibit the respiration of E. coli. This analogy should not be taken any further since the molecular
73
0.6
0.4
0.2
0.0 300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 3. UV–VIS spectra of orange II azo dye before and after the treatment with C. elegans UCP 542 on different media: control ( ), medium I ( ), medium II (––), medium III ( ) and medium IV ( ).
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S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75
the UV–VIS region were not significant and thus the dominant mechanism for decolorization would be the biosorption of the dye molecules (Zheng et al., 1999). Regarding the spectral shifts towards shorter wavelengths observed in the treatment with medias I and III, they were probably produced by the biodegradation of the dye molecule that led to a decrease of the conjugation effects between the aromatic rings. It should be noted that for conjugated systems the wavelength of the maximum absorption was very sensitive to the size of the conjugation, where a decrease of one unit of double bond can lead to shifts in range of 25–30 nm in polyeniccarbonyl conjugated systems (Streitwieser et al., 1992). In Fig. 4 we presented the UV–VIS spectra of the solution containing a mixture of all dyes before and after the fungal treatment. It can be readily observed that the decolorization that had already occurred just after 24 h is mainly due to biosorption, since there were no spectral differences between the control and the solution after the treatment. However, the solution after 96 h became re-colorized, due to some products from desorption, but mainly due to the degradation of the adsorbed dyes, since there were significant spectral shifts in the 500 and 400 nm regions. Thus, the results of the UV–VIS spectra corroborated the toxicity tests regarding the degradation of the dyes by C. elegans in the presence of sucrose. In addition, these data showed that it was viable to use UV–VIS spectroscopy to determine the kinetics of the biosorption and the degradation processes during the decolorization. In conclusion, the decolorization process produced by treatment with C. elegans was strongly dependent upon the co-metabolism conditions, where the sucrose enhanced and peptone had a negative effect upon the decolorization. This decolorization process was also dependent upon the molecular structure of the Mixture 0.6
Absorbance
0.5 0.4 Control 0.3 Mixture after 24 h 0.2
Mixture after 96 h
0.1 0.0 300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 4. UV–VIS spectra of the mixture of dyes, before ( ) and after 24 (- - -) and 96 ( ) h of treatment with C. elegans UCP 542 on condition medium III.
dye, despite the fact that the studied dyes had a common stilbene-azo structural motif. In addition, it has been shown by toxicity tests and UV–VIS spectral analysis that the decolorization process involved biodegradation, in addition to the visually observed biosorption.
Acknowledgements The authors gratefully acknowledge Prof. Dr. Ricardo L. Longo (DQF/UFPE) for assistance on analytical interpretation of the experimental data and review of the manuscript; Dra. Sandra G. Moraes (IQM/UNICAMP, SP) for the toxicity tests and Dra. Adriana M.Y. Melo (Pesquisadora CNPq/Embrapa rido) for helping with the statistical analyses. We Semi-A would like to acknowledge the Brazilian agencies CNPq, CAPES, the programs FINEP/CTPETRO/AVINA GROUP and PRONEX for financial support. References Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press. p. 302. Banat, I.M., Nigam, P., Singh, D., Marchant, R., 1996. Microbial decolorization of textile-dye containing effluents: a review. Bioresource Technology 58, 217–227. Bumpus, J.A., 1995. Microbial degradation of azo dyes. In: Singh, V.P. (Ed.), Biotransformations: Microbial degradation of healthrisk compounds. Elsevier Science, Amsterdam, pp. 157–175. Cha, C.J., Doerge, D.R., Cerniglia, C.E., 2001. Biotransformation of malachite green by fungus Cunninghamella elegans. Applied and Environmental Microbiology 67 (9), 4358–4360. Cripps, C., Bumpus, J.A., Aust, S.D., 1990. Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Applied Environmental Microbiology 56 (4), 1114–1118. Correia, V.M., Stephenson, T., Judd, S., 1994. Characterisation of textile wastewaters––a review. Environmental Technology 15, 917– 929. Duran, N., Esposito, E., 2000. Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Applied Catalysis B: Environmental 28, 83–99. Emtiazi, G., Satarii, M., Mazaherion, F., 2001. The utilization of aniline, chlorinated aniline, and aniline blue as the only source of nitrogen by fungi in water. Water Research 35 (5), 1219–1224. Fu, Y., Viraraghavan, T., 2001. Fungal decolorization of dye wastewaters: a review. Bioresource Technology 79, 251–262. Gomes, P.F., Nascimento, A.E., Okada, K., Messias, A.S., Sharia, A.E.N., Campos-Takaki, G.M., 2000. Aspectos da qualidade de ecossistemas do Municıpio do Rio Formoso. International Conference––Sustainable use of estuaries and mangroves: challenges and prospects, p. 82. Hansen, E.B., Cho, B.P., Korfmacher, W.A., Cerniglia, C.E., 1995. Fungal transformation of antihistamines: metabolism of brompheniramine, chlorpheniramine, and pheniramine to N-oxide and Ndemethylated metabolites by fungus Cunninghamella elegans. Xenobiotica 25 (10), 1081–1092. Hu, T.L., 2001. Kinetics of azoreductase and assessment of toxicity of metabolic products from azo dye by Pseudomonas luteola. Water Science and Technology 43 (2), 261–269. Kapdan, I.K., Kargi, F., McMullan, G., Marchant, R., 2000. Effect of environmental conditions on biological decolorization of textile
S.T. Ambrosio, G.M. Campos-Takaki / Bioresource Technology 91 (2004) 69–75 dye by C. versicolor. Enzyme and Microbial Technology 26, 381– 387. Kirby, N., Marchant, R., McMullan, G., 2000. Decolourisation of synthetic textile dyes by Phlebia tremellosa. FEMS Microbiology Letters 188, 93–96. Knapp, J.S., Newby, P.S., Reece, L.P., 1995. Decolorization of dyes by wood-rotting basidiomycete fungi. Enzyme and Microbial Technology 17, 664–668. Lacaz, C.S., Porto, E., Martins, J.E., 1991. Micologia Medica: fungos, actinomicetos e algas de interesse medico. 8th. S~ao Paulo: Savier, p. 695. Moraes, S.G., Freire, R.S., Duran, N., 2000. Degradation and toxicity reduction of textile effluent by combined photocatalytic and ozonation processes. Chemosphere 40, 369–373. asek, V., Molitoris, Novotn y, C., Rawal, B., Bhatt, M., Patel, M., S H.P., 2001. Capacity of Irpex lacteus and Pleurotus ostreaus for decolorization of chemically different dyes. Journal of Biotechnology 89, 113–122. Nyanhongo, G.S., Gomes, J., G€ uubitz, G.M., Zvauya, R., Read, J., Steiner, W., 2002. Decolorization of textile dyes by lacases from a newly isolated strain of Trametes modesta. Water Research 36, 1449–1456. OÕDonnell, K.L., 1979. Zygomycetes in Culture. Palfrey Contributions in Botany, no. 2. Department of Botany, University of Georgia, Athens, p. 257. Panswad, T., Luangdilok, W., 2000. Decolorization of reactive dyes with different molecular structures under different environmental conditions. Water Research 34 (17), 4177–4184. Robinson, T., McMullan, G., Marchant, R., Nigam, P., 2001. Remediation of dyes in textile effluent: a critical review on current
75
treatment technologies with a proposed alternative. Bioresource Technology 77, 247–255. Slockar, Y.M., Le Marechal, A.M., 1998. Methods of decoloration of textile wastewaters. Dye and Pigments 37 (4), 335–356. Statsoft Inc––Statistica for Windows. Computer Program Manual. Tulsa, 1997. Streitwieser, A.C.H., Heathcock, E., Kosower, M., 1992. Introduction to Organic Chemistry, fourth ed. Macmillan Publishing Co., New York. p. 673. Sumathi, S., Manju, B.S., 2000. Uptake of reactive textile dyes by Aspergillus foetidus. Enzyme and Microbial Technology 27, 347– 355. Tan, N.C.G., Lettinga, G.H., Field, J.A., 1999. Reduction of the azo dye mordant orange 1 by methanogenic granular sludge exposed to oxygen. Bioresource Technology 67, 35–42. Tatarko, M., Bumpus, J.A., 1998. Biodegradation of congo red by Phanerochaete chrysosporium. Water Research 32 (5), 1713–1717. Wackett, L.P., Gibson, D., 1982. Metabolism of xenobiot compounds by enzymes in cell extracts of the fungus Cunninghamella elegans. Biochemical Journal 205, 117–122. Wang, Y., Yu, J., 1998. Adsorption and degradation of synthetic dyes on the mycelium of Trametes versicolor. Water Science Technology 38 (4–5), 233–238. Zhang, D., Yang, Y., Leakey, J.E.A., Cerniglia, C.E., 1996. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiology Letters 138, 221– 226. Zheng, Z.X., Levin, R.E., Pinkham, J.L., Shetty, K., 1999. Decolorization of polymeric dyes by a novel Penicillium isolate. Process Biochemistry 34 (1), 31–37.