Degradation of azo dyes by oxidative processes – Laccase and ultrasound treatment

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 4213–4220

Degradation of azo dyes by oxidative processes – Laccase and ultrasound treatment Michael M. Tauber

a,b

, Georg M. Gu¨bitz b, Astrid Rehorek

a,*

a

b

University of Applied Sciences Cologne, Faculty of Process Engineering, Energy and Mechanical Systems, Department of Chemical Engineering and Plant Design, Betzdorfer Straße 2, D-50679 Cologne, Germany University of Technology Graz, Institute for Environmental Biotechnology, Petersgasse 10, A-8010 Graz, Austria Received 21 July 2004; received in revised form 6 February 2007; accepted 28 August 2007 Available online 29 October 2007

Abstract Azo dyes are of synthetic origin and their environmental fate is not well understood. They are resistant to direct aerobic bacterial degradation and form potentially carcinogenic aromatic amines by reduction of the azo group. This study shows that applying the oxidative processes of enzymatic treatment with laccase and ultrasound treatment, both alone and in combination, leads to dye degradation. Laccase treatment degraded both Acid Orange and Direct Blue dyes within 1–5 h but failed in the case of Reactive dyes, whereas ultrasound degraded all the dyes investigated (3–15 h). When applied as multi-stage combinations the treatments showed synergistic effects for dye degradation compared with individual treatments. Bulk light absorption (UV–Vis) and ion pairing HPLC were used for process monitoring. Additionally, mass spectrometry was used to elucidate the structures of intermediates arising from ultrasound treatment.  2007 Elsevier Ltd. All rights reserved. Keywords: Azo dyes; Laccase; Ultrasound; Ion pairing HPLC; Mass spectrometry

1. Introduction Dye-house effluents contain large amounts of dyes. Sulfonylester groups of unbound reactive dyes undergo hydrolysis due to elevated temperature and pH value during dyeing processes. The discharged dyes, even at very small concentrations, have a high impact on the aquatic environment due to BOD, colour and turbidity (Lin and Lin, 1993). Additionally, toxic degradation products can be formed. Azo dyes constitute the largest group of colorants used in industry and they pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment. Little is known about their environmental fate. Under anaerobic conditions, azo dyes are cleaved by micro-organisms forming potentially carcinogenic *

Corresponding author. Tel.: +49 221 8275 2234; fax: +49 221 8275 2202. E-mail address: [email protected] (A. Rehorek). 0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.08.085

aromatic amines (Chung and Cerniglia, 1992). This can occur in river sediments. The fragments arising from the azo bond cleavage can undergo autoxidation under aerobic conditions, forming colored products; this could take place in rivers. For instance, CI Acid Orange 52 forms ‘‘aniline black’’ by polymerization of anaerobically formed fragments (Christen and Vo¨gtle, 1989). Azo dyes forming ‘‘forbidden aromatic amines’’ are banned from production in Germany. Among these forbidden aromatic amines there are some alkylated derivatives of aniline such as 2,4,5-trimethylaniline and o-toluidine, naphthylamine derivatives such as 2-naphthylamine and benzidine derivatives such as aminobiphenyls (Bedarfsgegensta¨ndeverordnung, 1998). They are toxic and potential carcinogens (Chung and Cerniglia, 1992), which possibly form toxic metabolites after hydroxylation or oxidation with cytochrome P450 inside mammalian cells (Sterner, 1999). All these mechanisms can take place after release from waste water plants, creating an urgent demand for the development of multi-step treatment processes

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which not only guarantee irreversible decolorization but also nearly complete mineralization of the dyes into water, nitrogen, sulfate and CO2. For the study presented here, laccase from the white rot fungus Trametes modesta was chosen. Laccases are well known as benzenediol:oxygen oxidoreductases (EC 1.10.3.2) and belong to the class of blue oxidases. Their typical molecular mass ranges between 60 and 85 kDa (Calvo et al., 1998; Wahleithner et al., 1996). Laccases are capable of catalyzing a 4-electron-transfer reaction necessary to reduce molecular oxygen, which is used as the terminal electron acceptor, thus forming water without the formation of H2O2. Laccases have a very broad substrate specificity with respect to the electron donor. These enzymes catalyze the removal of a hydrogen atom from the hydroxyl group of ortho and para substituted monoand poly-phenolic substrates and from aromatic amines by one electron abstraction to form free radicals capable of undergoing polymerization to form phenolic polymers (Bollag, 1992). Further electron removal will lead to depolymerization, repolymerization, demethylation or quinone formation (Goncalves and Steiner, 1996). Although laccases have been shown to degrade textile dyes (Kandelbauer et al., 2006; Nyanhongo et al., 2002b; Soares et al., 2002; Wong and Yu, 1999), the majority of dyes are only degraded in the presence of laccase mediators. However, since most of these mediators are toxic and/or expensive, the potential of ultrasound to extend substrate range and decolorization efficiency in enzymatic dye degradation was studied in the present paper. Ultrasound has found widespread industrial applications, such as surface treatment, soldering and formation of emulsions (Suslick, 1988). Its application in organic chemistry has also become increasingly important, especially for the synthesis of organometallic compounds (Ley and Low, 1989). When aqueous solutions are exposed to ultrasound, transient cavitations are formed due to compression and rarefaction of the bulk water. The cavitations collapse locally producing high pressure and temperature

peaks (500 bar, 5000 K). Under these extreme conditions hydroxyl radicals and hydrogen atoms are formed by opening the H–O bond (Suslick, 1988). Volatile and hydrophilic compounds react at the layer between cavitation and bulk water with the supercritical water and inside the bulk water with the ejected hydroxyl radicals, while simple volatile and hydrophobic compounds are pyrolyzed inside the cavitation bubble (Hua et al., 1995). Recent results confirm the potential of pulsed sonolysis (Casadonte et al., 2005). Ultrasonication of azo dyes possibly leads to nitro and nitroso aromatics (Joseph et al., 2000), whose acute and chronic toxicity and carcinogenic properties result in a high pollution potential (Rickert, 1987; Schwedt, 1996). Therefore, it is of outstanding interest to find a combination with another treatment such as enzymic degradation with laccases to enhance overall dye degradation while avoiding the formation of toxic degradation products. 2. Methods 2.1. Chemicals Six azo dyes were investigated (Fig. 1): CI Acid Orange 5 (Aldrich), CI Acid Orange 52 (Merck), CI Direct Blue 71 (Aldrich), CI Reactive Black 5 (DyStar), CI Reactive Orange 16 (DyStar) and CI Reactive Orange 107 (DyStar). All other chemicals used (p.a. quality) were from Sigma and the HPLC solvents used in gradient grade quality were from Merck, Germany. All three reactive dyes were hydrolyzed at 70 C and pH 12 for 24 h. Crystallization in dry methanol was used for purification. All experiments were performed at 100 lM initial dye concentration. 2.2. Laccase production and assay Laccase from T. modesta was produced as reported previously (Nyanhongo et al., 2002a) and was precipitated from a culture filtrate with (NH4)2SO4 at 70% w/w satura-

SO3Na SO3Na

NaO3S N

N

N N H

1

N

N

N

SO3Na N

2

N

CH3

OH O2S

OH N

N

SO2

NH2 N

NaO3S

N

N

NaO3S

NH2

3

SO Na

HO

HO

O2S

O2S

OH

N

SO3Na

4

OH

CH3

3 HO

N

N

5

N

NaO3S

H N

COCH 3

HN N

6

COCH3

N

NH2 SO3Na

Fig. 1. Structures of the azo dyes employed: (1) CI Acid Orange 5, (2) CI Acid Orange 52, (3) CI Direct Blue 71, (4) hydrolyzed CI Reactive Black 5, (5) hydrolyzed CI Reactive Orange 16, (6) hydrolyzed CI Reactive Orange 107.

M.M. Tauber et al. / Bioresource Technology 99 (2008) 4213–4220

tion at room temperature. The laccase was used without further purification steps. Laccase activity was determined by monitoring the formation of the dimeric oxidation product from 2,6-dimethoxyphenol as described previously (Benefield et al., 1964).

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following conditions: 300 nl/min, 800 V, 0.3 lA, inlet temperature 230 C. 3. Results 3.1. Enzymatic treatment, UV–Vis detection

2.3. Dye degradation studies The laccase treatment was operated at 40 C, pH 4.5, acetate buffer 50 mM and 5 · 10 9 kat/ml. Reactions were stopped by addition of 10 mM NaF (final concentration) (Xu et al., 1996) and samples were kept frozen until further preparation and analysis. Ultrasound treatment was performed with an ultrasound device K 80 (Meinhardt Ultraschalltechnik, Leipzig, Germany) at 850 kHz, 90 W (4.1 W cm 2) and 30 C (303 K) in continuous operation mode in a stirred batch with a cooling device for temperature control. Sequential treatment was carried out first with laccase and followed by the ultrasound treatment under the conditions described above. Simultaneous treatment was carried out at 40 C and pH 4.5. UV–Vis spectrometry was performed with a Perkin Elmer, Lambda 10 spectrophotometer (USA). The spectra were recorded between 200 and 800 nm at a scan rate of 240 nm min 1. HPLC analysis was carried out with the LaChrom System, Merck HITACHI, DAD L-7450A (200–800 nm), RP-select B 5 lM 125 · 4 LiChroshere 60 column (Merck KgaA, Germany). Eluent A: 1 mM tetrabutylammonium bisulfate (TBAHS) in water plus 10% v/v CH3CN; eluent B: CH3CN. Gradient profile: 0–5 min 100% A, 5– 30 min linear gradient +2% min 1 B, 30–34 min linear gradient +10% min 1 B, 34–36 min 90% B, 36 min 37 min return to 0% B, 37–45 min 0% B. Eluent flow rate 1 ml min 1. Samples were centrifuged at 10,000g for 10 min to remove any suspended particles. Mass spectroscopy (MS) was operated as nano electron spectroscopy (ESI) with a Thermo Finnigan MAT and the

Various azo dyes were decolorized by the laccase from T. modesta at different rates (first order), as shown in Table 1. Acid Orange 52 and Direct Blue 71 showed fastest decolorization (more than 50% decolorization after 2 h), while both CI Reactive Orange 16 and CI Reactive Orange 107 were not decolorized at all within 70 h of incubation. 3.2. Enzymatic treatment, HPLC analysis CI Acid Orange 5 formed four intermediates (Fig. 2) with one showing a higher retention time than the parent peak. CI Acid Orange 52 also formed four intermediates. After 2 h both Acid Orange dyes were nearly completely degraded, while CI Direct Blue 71 showed a residual dye concentration of 10 lM and the formation of several intermediates in the same amount of time. The intermediates with faster retention times absorbed in the UV region and those with slower retention times absorbed in the visible region. Both Acid Orange dyes and Direct Blue 71 presented high degradation rates (Table 1). CI Reactive Black 5 formed one intermediate (Fig. 2) by laccase treatment and no degradation of either Reactive Orange dye was detectable. CI Direct Blue 71 initially displayed first order degradation, which changed to a higher order after 15 min at 5 · 10 9 kat/ml. Repeated addition of dye, conducted at 1 · 10 9 kat/ml laccase activity, was used to investigate the potential occurrence of inhibiting intermediates. After 80% decolorization, the same amount of dye was added and the subsequent decolorization showed the same kinetic rate, as indicated in Fig. 3. The formation rates of intermediates were also reproducible.

Table 1 survey of k-values (first order, 1/h) for laccase and ultrasound treatment at 100 lM initial dye concentration and 5 nkat/ml if not otherwise stated Laccase

Ultrasound

Combination

UV–Vis

HPLC

UV–Vis

HPLC

Sequential UV–Vis

Simultaneous HPLC

CI Acid Orange 5 CI Acid Orange 52

0.24 ± 0.02 0.36 ± 0.02

0.97 ± 0.10 1.82 ± 0.37

1.21 ± 0.43 1.66 ± 0.03

No values No values

1.73 ± 0.12 2.39 ± 0.21

CI Direct Blue 71 CI Reactive Black 5

1.7 ± 0.1 0.006 ± 0.003

4.96 ± 0.30 0.03 ± 0.00

0.30 ± 0.11 0.34 ± 0.05

No values No values

7.18 ± 1.05 0.30 ± 0.04

CI Reactive Orange 16

0.00 ± 0.00

0.00 ± 0.00

1.6 (54 lM) 0.67 ± 0.07 68a (200 lM) 75 ± 2a (300 lM) 24a 0.53 (59 lM) 46a (550 lM) n.d.

0.44 ± 0.08

No values

0.45 ± 0.03

CI Reactive Orange 107

0.00 ± 0.00

0.00 ± 0.00

n.d.

0.84 ± 0.43

No values

1.29 ± 0.11

Mean values and standard deviation (n = 3). a Zeroth order (lM h 1)

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M.M. Tauber et al. / Bioresource Technology 99 (2008) 4213–4220 Dyes AcO5

387

AcO52

337 386

DBl71

420

319

420

444 435

417

227 235

RBl5 RO16

440

561 570 444 555 560 510 515 567

555

20

25

484

RO107

444 15

30

Retention Time [min]

Fig. 2. Intermediates formed with laccase obtained by HPLC. Dye peaks are in bold numbers.

Reactive Orange 16 showed nearly complete decolorization after 23 h. CI Reactive Orange 107 exhibited a moderate decolorization rate. 3.4. Ultrasound treatment, HPLC analysis

Fig. 3. Double addition of Direct Blue 71 at 1 nkat/ml. After 60 min the dye was added a second time.

Concordant HPLC and UV–Vis data indicated good or excellent degradation of CI Acid Orange 5, 52 and CI Direct Blue 71 by laccases and poor or no degradation of the reactive dyes. The comparison between HPLC and UV–Vis data indicated that formation of one peak in the UV–Vis spectra corresponded to several peaks observed by HPLC. Additionally, UV–Vis showed a very large blank absorption below 220 nm, making it impossible to detect intermediates qualitatively in this region. 3.3. Ultrasound treatment, UV–Vis detection After 1.3 h of ultrasound treatment CI Acid Orange 5 was almost completely degraded. At 220 nm and 330 nm, absorption increases were detectable. No formation of intermediates was discovered for CI Acid Orange 52. CI Direct Blue 71 underwent complete decolorization after 23 h. 80% of CI Reactive Black 5 was decolorized after 3.5 h, with complete decolorization achieved after 9 h. CI Dyes AcO5 AcO52 DBl71 RBl5H RO16H RO107H

279 444 15

Both Acid Orange dyes showed the formation of at least five colored degradation products absorbing between 320 and 470 nm (Fig. 4), which were completely degraded after 3 h of treatment. CI Direct Blue 71 and CI Reactive Black 5 showed the slowest degradation (Table 1). With CI Direct Blue 71, CI Reactive Black 5 and CI Reactive Orange 16 only three, two and one intermediates were formed, respectively. No intermediates were detected in the case of CI Reactive Orange 107. 3.5. Ultrasound treatment, MS data MS analysis of CI Acid Orange 52 after 1 h of ultrasonication showed the following m/z values for the negative nano ESI mode (in parenthesis the most probable structure): 138 (O2N–Ph@O , quinoic structure), 157 ( O3S– Ph*, radical anion), 173 ( O3S–Ph–OH), 241, 260 (M– NMe2 , radical anion), 277 (M 2 · CH2+H , radical anion), 290 (M CH2) 304 (AcO52–Na = M), 320 (M+O) , 336 (M+2O) , 352, 368 (condensation products). m/z at 327, corresponding to the unchanged molecular peak, was not detected. 3.6. Combination of laccase and ultrasound treatment Since both laccase and ultrasound treatment degraded only selected dyes efficiently, the effect of a combination of these methods was investigated. In the case of sequential

420 430 420 441 420 354 319 420 342 444 468 373 228 561 570 561 510 350 567 573 555 484 20

25

30

Retention Time [min]

Fig. 4. Intermediates formed by ultrasound treatment obtained by HPLC. Dye peaks are in bold numbers.

M.M. Tauber et al. / Bioresource Technology 99 (2008) 4213–4220

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Dyes 391

AcO5

341

420 420 400

366 350 340 419 344 444

AcO52

515 290 230 235

DBl71

405

530 570

513 514 567 550

RBl5H

541 550

553

484

RO16H 444

RO107H 15

20

25

30

Retention Time [min]

Fig. 5. Intermediates formed by the simultaneous combination method obtained by HPLC. Dye peaks are in bold numbers.

treatment the substrates were treated with laccase first and then irradiated with ultrasound. Fig. 6 shows the UV–Vis spectra. CI Acid Orange 5 and 52 showed good enzymatic decolorization and were completely decolorized by ultrasound. The sequential combination did not give the same result as ultrasound alone, which can be seen from the formation of different intermediates. CI Direct Blue 71 was degraded by about 90% by laccase, while ultrasound treatment led to a complete decolorization after 23 h. Complete decolorization was also achieved by a combination of these methods. The enzymatic degradation of CI Reactive Black 5 was difficult to accomplish. However, it could be decolorized by ultrasound after 9 h. The combination showed a final concentration of 20%, which is a remarkable improvement but no advantage compared with ultrasound alone. CI Reactive Orange 16 and 107 showed the smallest k-values in the laccase treatment and moderate degradation with ultrasound, but the combination showed the best improvement in the case of CI Reactive Orange 107. A further approach in applying a combination method was the simultaneous action of laccase and ultrasound treatments. It was proved that laccase was deactivated by only 7% during the first two hours of low frequency ultrasound treatment. Calculating data from Fig. 5, a deactivation half time of around 20 h (k  3.7 · 10 2 h 1) can be derived under these conditions, which is as high as values obtained by native deactivation (k  3.3 · 10 2 h 1) (Nyanhongo et al., 2002a). The observed intermediates are shown in Fig. 5 and the kinetic data for the simultaneous treatment are shown in Table 1. As a minimum, in all cases the simultaneous treatment gave degradation rates as high as those in the individual treatments. Both Acid Orange dyes showed slightly higher values. CI Direct Blue 71 and CI Reactive Orange 107 showed increases higher than would be predicted by the simple addition of results from the two treatments. 4. Discussion In this study, it is shown that CI Acid Orange 5, 52 and CI Direct Blue 71 were degraded by a laccase from T. modesta, while reactive dyes seemed to be resistant. In contrast to several studies on dye degradation (Peralta-Zamora et al., 2001; Rodrı´guez et al., 1999; Soares et al., 2002;

Wong and Yu, 1999), HPLC analysis in addition to UV/ Vis spectroscopy was used to monitor the reaction. Usually, waste water is monitored by UV–Vis spectroscopy according to DIN EN ISO 7887 at three different wavelengths (436, 525 and 620 nm). This method offers only a rough estimation of the absorption decreases in the visible region without any possibility of obtaining further information about degradation products and intermediates. In this study, it is clearly shown that UV/Vis spectroscopy alone can lead to misinterpretation of the actual degradation process, because intermediates often absorb around the maximum wavelength of the parent azo dye (Figs. 2, 4 and 5) and simulate higher absorbances and lower degradation rates, which is also valid for ultrasound treatment. HPLC offers the possibility of obtaining more detailed data. Additionally, HPLC on-line and in-line application provides the advantage of monitoring changes, lying in the time scale of minutes or hours, directly in bioreactor systems and waste water treatment units (Rehorek et al., 2002; Plum et al., 2003). The rate of degradation of CI Acid Orange 52 by laccase was nearly 2-fold higher than that of CI Acid Orange 5, which can be explained by the higher electron-donating properties of the two methyl groups present compared with phenylic systems (CI Acid Orange 5). Previously, dyes with electron-donating substituents such as methyl or methoxy groups have been reported to give the highest degradation rates, suggesting that only an electron rich phenolic ring can be oxidized by laccase (Chivukula and Renganathan, 1995). Garzillo et al. (1998) reported that, for a Trametes trogii laccase, introduction of o- or p-orienting ring-activation groups may convert recalcitrant phenolic molecules into substrates by increasing the electron density at the phenolic hydroxyl group. Additionally, the substituent location plays an important role, shown by the overwhelmingly higher degradation rates of 2,6-, 2,3- and 2-phenol substituents compared with 3- and 3,5-analogs (Chivukula and Renganathan, 1995). The enzymatic degradation of azo dyes with fungal laccase has been found to lead to the formation of quinones and hydroperoxides under nitrogen loss (Chivukula and Renganathan, 1995; Heinfling et al., 1997; Rodrı´guez et al., 1999; Schliephake et al., 2000). The treatment of CI Acid Orange 5 and 52 by laccases should come to a halt after the formation of hydroquinones,

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because their intermediates lack –OH, –NH2, –NHR or –NR2 groups for further enzymatic attacks. However, CI Acid Orange 5, CI Acid Orange 52 and CI Direct Blue 71 showed the highest degradation rates and very pronounced product formation as detected by HPLC (Fig. 6). While intermediates formed by CI Acid Orange 5 and 52 also disappeared, CI Direct Blue 71 showed at least two reaction end products (227 and 235 nm). The lack of these peaks, especially in the case of CI Reactive Black 5, suggests, on the one hand, that these fragments are degraded very quickly, because they still possess –OH, –NHR, groups, which can be attacked once again by laccase. On the other hand, these fragments may possess absorption maxima below the HPLC detection limit of 200 nm. Repeated addition of CI Direct Blue 71 showed identical pseudo first order degradation rates and reproducible formation of products. This observation excludes the possibility of laccase inhibition by these products which may still possess –OH, –NH2 groups, and therefore could lead to competition with the parent dye molecule. The findings suggest that the high degradation rates observed with laccase (apart from CI Acid Orange 5 and 52) correlate with the easy accessibility of the amine groups (Fig. 1). CI Reactive Black 5 was hardly decolorized. This dye undergoes tautomerization to a ketohydrazine derivative (Pham et al., 2001), which is also possible for CI Reac-

tive Orange 16, leading to restricted accessibility of the amino group due to steric hindrance. Both Acid Orange dyes and their intermediates were degraded completely. These observations may be explained by the broad substrate specificity of laccase or the possible action of internal mediators, being intermediates formed after the first laccase attack. In addition to degradation of phenolic compounds, laccase can also cause polymerization through radical coupling and depolymerization (Bollag et al., 1988; Chivukula and Renganathan, 1995). Radical coupling takes place in the ortho or para-position of the phenolic compound; if these positions were blocked by methyl or methoxy substituents, polymerization could be reduced or eliminated. In this study, no precipitation was observed during enzymatic treatment of dyes. Experimental data for CI Acid Orange 52 and CI Direct Blue 71 ultrasound degradation showed a zero order decolorization at higher initial dye concentration. Further concentration increases will not increase the degradation rates. Low initial dye concentrations led to a pseudo 1st order rate decolorization (Table 1). These findings may be explained by a constant supply of OH-radicals (Hua and Hoffmann, 1997). At low initial dye concentrations the probability of an OH-radical attack on a dye molecule is proportional to the dye’s concentration, which mathe-

3

abs

abs

2

0 200

500 nm

0 200

800

Acid Orange 5

500 nm

800

Reactive Black 5 2

abs

abs

2

0 200

500 nm

0 200

800

Acid Orange 52

500 nm

800

Reactive Orange 16

2

abs

abs

2

0 200

500 nm

Direct Blue 71

800

0 200

500 nm

800

Reactive Orange 107

Fig. 6. Sequential combination treatment – before treatment (bold line), 24 h laccase (fine line), 24 h laccase plus 18–25 h ultrasound (dotted line), c0  50 lM.

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matically defines the first order degradation. As the reaction proceeds, degradation velocities decrease independently of the initial dye concentration. The enrichment of low molecular weight species (Stock et al., 2000) is responsible for this observation, because they are more difficult to degrade and may act as quenchers. Independent investigations show that the total organic carbon (TOC) decreases to a residual concentration of around 20% for CI Reactive Black 5, which was assumed to be oxalate (Vinodgopal et al., 1998). In the case of CI Acid Orange 52, 50% residual TOC concentration was found (Joseph et al., 2000), which confirms the statement above. The same research group found, under conditions similar to those in the present study (500 kHz, 50 W, 288 K, c0 = 10 lM), a pseudo first order constant of 2.4–3.0 h 1 for CI Acid Orange 52. Observations in the present study revealed a constant of 1.5–2.2 h 1 (c0 = 100 lM). Vinodgopal et al. (1998) described the attack of non-volatile Reactive Black 5 in the ‘‘bulk’’ water by OH radicals, which destroyed the chromophoric system through azo bond cleavage. Joseph et al. (2000) assumed that the OH attack leads to hydroxyl amines (which were not detected by mass spectrometry analysis), followed by a subsequent oxidation leading to nitroso and nitro aromatic compounds. Different mechanisms, including the attack of the azo link bearing carbon, leading to phenyl derivative radicals have been postulated (Galindo et al., 2000; Rasanu et al., 2000), which presents difficulties in describing the first step of this degradation process qualitatively. Mass spectrometry results in the present work confirm the proposed mechanism of a direct azo bond attack (m/z: 138) (Joseph et al., 2000). Strong evidence was also found for the attack of the azo bond bearing carbon atom leading to m/z: 157, 173 (Galindo et al., 2000). Furthermore, sequential demethylation was observed (m/z: 290, 277), followed by radical formation (m/z: 260). During simultaneous treatment with laccase and ultrasound, both CI Acid Orange dyes showed slightly higher degradation rates than those observed after application of either of the single methods alone, but CI Direct Blue 71 and CI Reactive Orange 107 displayed the most pronounced increases. For the latter two dyes, ultrasonication possibly creates intermediates acting as internal mediators for laccase. Mediators are believed to oxidize non-substrate molecules or to enhance degradation rates by undergoing a redox cycle between laccase enzyme and the target molecule (Johannes et al., 1996; Johannes and Majcherczyk, 2000; Nyanhongo et al., 2002b). The small k-value increase and the lower concentration of intermediates in the case of both CI Acid Orange dyes may have been due to the competition for and capture of the hydroxyl radicals by these intermediates. No benefit was observed with CI Reactive Black 5 and CI Reactive Orange 16. The lack of intermediates in the chromatograms should not be interpreted as absence of intermediates but could possibly be due to the formation of intermediates that absorb below 200 nm.

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The observed features of intermediates were not the same as those after the individual treatments. Some intermediates did not occur, while new ones could be observed (Figs. 2, 4 and 5), which was very pronounced in the case of CI Direct Blue 71. Laccase deactivation during the first two hours of simultaneous treatment was similar to native deactivation (Nyanhongo et al., 2002a) under the same conditions without ultrasonication. Therefore, the laccase was not deactivated by ultrasound of higher frequency. In particular, these findings indicate the feasibility of a new combination of methods including ultrasound and enzymatic treatment. 5. Conclusions Laccase enzyme treatment and ultrasonication are two oxidizing processes, which are able to degrade azo dyes generally but to differing extents. In the case of enzymatic treatment, CI Acid Orange 52 and CI Direct Blue 71 showed the highest decolorization rates. Complete decolorization of all dyes investigated was achieved with the ultrasound treatment. The sequential combination of laccase and ultrasound treatment was in most cases an improvement compared with laccase treatment without ultrasound. CI Direct Blue 71 was completely decolorized, which was also achieved by ultrasound treatment alone. CI Reactive Black 5 showed a better decolorization compared with the enzyme treatment alone, but a worse decolorization compared with the ultrasound step. CI Reactive Orange 107, resistant against laccase treatment, was completely degraded by the sequential combination method, which is a tremendous improvement compared with the single treatment steps. The simultaneous combination of laccase and ultrasound treatment showed a synergistic effect on degradation rates in two cases (CI Direct Blue 71 and CI Reactive Orange 107). For the first time, it has been shown that a simultaneous treatment with laccase and ultrasound could save time and energy, without the laccase performance being affected by use of ultrasound. Ongoing investigations will focus on the structural investigation of the intermediates formed by laccase and ultrasound to elucidate the degradation mechanisms. Acknowledgement We would like to thank Prof. M. Scha¨fer, University of Cologne, Institute of Mass Spectroscopy Facilities, for the mass spectrometry measurements. References Bedarfsgegensta¨ndeverordnung, 1998. Fassung 23. Dezember 1997, Bundesgesetzblatt I. Benefield, G., Bocks, S.M., Bromley, K., Brown, B.R., 1964. Studies of fungal and plant laccases. Phytochemistry 3, 79–88.

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Bollag, J.M., 1992. Enzymes catalyzing oxidative coupling reactions of pollutants. Metal Ions Biol. Syst. 28, 205–217. Bollag, J.M., Shuttleworth, K.L., Anderson, D.H., 1988. Laccase mediated detoxification of phenolic compounds. Appl. Environ. Microbiol. 54 (12), 3086–3091. Calvo, A.M., Copa-Patino, J.L., Alonso, O., Gonza´lez, A.E., 1998. Studies of the production and characterization of laccase activity in the basidiomycete Coriolopsis gallica, an efficient decolorizer. Arch. Microbiol. 171, 31–36. Casadonte Jr., D.J., Flores, M., Petrier, C., 2005. Enhancing sonochemical activity in aqueous media using power-modulated pulsed ultrasound: an initial study. Ultrason. Sonochem. 12, 147–152. Chivukula, M., Renganathan, V., 1995. Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl. Environ. Microbiol. 34 (23), 4374–4377. Christen, H.R., Vo¨gtle, F., 1989. Grundlagen der Organischen Chemie. Salle und Sauerla¨nder Verlag, Frankfurt am Main, Germany, pp. 566. Chung, K.-T., Cerniglia, C.E., 1992. Mutagenicity of azo dyes: Structure– activity relationships. Mutat. Res. 227, 201–220. Galindo, C., Jaques, P., Kalt, A., 2000. Photodegradation of the aminobenzene Acid Orange 52 by three AOPs: UV/H2O2, UV/TiO2 and VIS/TiO2. Comparative mechanistic and kinetic investigations. J. Photochem. Photobiol. A: Chem. 130, 35–47. Garzillo, A.M.V., Colao, M.C., Caruso, C., Caporale, C., Celletti, D., Buonocore, V., 1998. Laccase from white-rot fungus Trametes trogii. Appl. Microbiol. Biotechnol. 49, 545–551. Goncalves, M.L., Steiner, W., 1996. Purification and characterisation of laccase from a newly isolated wood decaying fungus. ACS Symp. Ser. 655, 258–266. Heinfling, A., Bergbauer, M., Szewzyk, U., 1997. Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta. Appl. Microbiol. Biotechnol. 48, 261–266. Hua, I., Ho¨chemer, R.H., Hoffmann, M.R., 1995. Sonolytic hydrolysis of p-nitrophenyl acetate: the role of supercritical water. J. Phys. Chem. 99, 2335–2342. Hua, I., Hoffmann, M.R., 1997. Optimization of ultrasonic irradiation as an advanced oxidation technology. Environ. Sci. Technol. 31, 2237– 2243. Johannes, C., Majcherczyk, A., 2000. Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl. Environ. Microbiol. 66 (2), 524–528. Johannes, C., Majcherczyk, A., Hu¨ttermann, A., 1996. Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds. Appl. Microbiol. Biotechnol. 46, 313–317. Joseph, J.M., Destaillats, H., Hung, H.-M., Hoffmann, M.R., 2000. The sonochemical degradation of azobenzene and related azo dyes: rate enhancements via Fenton’s reactions. J. Phys. Chem. A 104, 301–307. Kandelbauer, A., Maute, O., Kimmig, M., Kessler, R., Guebitz, G.M., 2006. Laccase catalyzed indigo carmine transformation. J. Nat. Fibers 3 (2/3), 131–153. Ley, St.V., Low, C.M.R., 1989. Ultrasound in Synthesis. Springer-Verlag, Berlin, Germany, ISBN 3-540-51023-0. Lin, S.H., Lin, C.M., 1993. Treatment of textile waste effluents by ozonisation and chemical coagulation. Water Res. 27, 1743–1748. Nyanhongo, G.S., Gomes, J., Gu¨bitz, G.M., Zvauya, R., Read, J., Steiner, W., 2002a. Optimisation of some variables for the production of laccase from a newly isolated strain of Trametes modesta. Bioresource Technol. 84 (3), 259–263.

Nyanhongo, G.S., Gomes, J., Gu¨bitz, G.M., Zvauya, R., Read, J., Steiner, W., 2002b. Decolorization of textile dyes by laccase from a newly isolated strain of Trametes modesta. Water Res. 36 (6), 1449– 1456. Peralta-Zamora, P., Tiburtius, E.R.L., Moares, S.G.d, Dura´n, N., 2001. Degradac¸a˜o enzima´tica de corantes texteis. In: Semina´rio International Aplicac¸a˜o da biotecnologia na Indu´stria Teˆxtil, Universidade Regional de Blumenau, Brazil, pp. 145–153. Pham, T.L.H., Rotard, W., Preiss, A., Elend, M., 2001. Mo¨glichkeiten und Grenzen der LC–MS Analyse von Farbstoffmetaboliten. SFB 193, Biologische Abwasserreinigung, Kolloquium an der TU Berlin – oral presentation/hand out. Plum, A., Braun, G., Rehorek, A., 2003. Process monitoring of anaerobic azo dye degradation by high-performance liquid chromatography– diode array detection continuously coupled to membrane filtration sampling modules. J. Chromatogr. A 987, 395–402. Rasanu, N., Chirila, E., Marza, V., Dobrinas, S., 2000. Descompunerea unor coloranti disaoici in camp ultrasonor. Rev. Chim. 51 (5), 349– 353. Rehorek, A., Urbig, K., Meurer, R., Scha¨fer, C., Plum, A., Braun, G., 2002. Monitoring of azo dye degradation processes in a bioreactor by on-line high-performance liquid chromatography. J. Chromatogr. A 949, 263–268. Rickert, D.E., 1987. Metabolism of nitroaromatic compounds. Drug Metab. Rev. 18, 23–53. Rodrı´guez, E., Pickard, M.A., Vazquez-Duhalt, R., 1999. Industrial dye decolorization by laccases from ligninolytic fungi. Curr. Microbiol. 38, 27–32. Schliephake, K., Mainwaring, D.E., Lonergan, G.T., Jones, I.K., Baker, W.L., 2000. Transformation and degradation of the diazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus. Enzyme Microb. Technol. 27, 100–107. Schwedt, G. Toxikologisches Lexikon. S.72. Vogel Verlag, Wu¨rzburg, ISBN: 3-8023-1569-3. Soares, G.M.B., Amorim, M.T.P., Hrdina, R., Costa-Ferreira, M., 2002. Studies on the biotransformation of novel azo dyes by laccase. Process Biochem. 37, 581–587. Sterner, O., 1999. Chemistry, Health and Environment. Wiley-VCH, Wreinheim, Germany. Stock, N., Peller, J., Vinodgopal, K., Kamat, P.V., 2000. Combinative sonolysis and photocatalysis for textile dye degradation. Environ. Sci. Technol. 34, 1747–1750. Suslick, K.S. (Ed.), 1988. Ultrasound. VCH, Weinheim, Germany, pp. 123–163 (Chapter 4). Vinodgopal, K., Peller, J., Makogon, O., Kamat, P.V., Vinodgopal, K., 1998. Ultrasonic mineralization of a reactive textile azo dye, Remazol Black B. Water Res. 32 (12), 3646–3650. Wahleithner, J.A., Xu, F., Brown, K.M., Brown, St.H., Golightly, E.J., Halkier, T., Kauppinen, S., Pedrson, A., Schneider, P., 1996. The identification and characterisation of four laccases from the plant pathogenic fungus Rhizoctonia solani. Curr. Genet. 29, 395–403. Wong, Y., Yu, J., 1999. Laccase-catalysed decolorization of synthetic dyes. Water Res. 33 (16), 3512–3520. Xu, F., Shin, W., Brown, S.H., Wahleithner, J.A., Sundaram, U.M., Solomon, E.I., 1996. A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. Biochim. Biophys. Acta 1292, 303–311.