APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2005, p. 2600–2607 0099-2240/05/$08.00 0 doi:10.1128/AEM.71.5.2600–2607.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Vol. 71, No. 5 Degradation of Azo Dyes by Laccase and Ultrasound Treatment Michael M. Tauber,1,2 Georg M. Guebitz,2 and Astrid Rehorek1* University of Applied Sciences Cologne, Chemical Engineering and Plant Design, Betzdorfer Strasse 2, D-50679 Cologne, Germany,1 and Graz University of Technology, Institute for Environmental Biotechnology, Petersgasse 12, A-8010 Graz, Austria2 Received 13 June 2004/Accepted 21 November 2004 The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversedphase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatographymass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on ˙OH attack on the dye molecules. Dye house effluents contain large amounts of dyes. Unbound reactive dyes undergo hydrolysis due to elevated temperature and pH values during the dyeing processes. The strong color of discharged dyes even at very small concentrations has a huge impact on the aquatic environment caused by its turbidity and high pollution strength (30). Additionally, toxic degradation products can be formed. Azo dyes, which constitute the largest group of colorants used in industry (57), leave municipal wastewater plants highly diluted but nearly unchanged because they resist aerobic and short-term anaerobic treatment (44, 51). Only small amounts can be precipitated or adsorbed, while under anaerobic conditions azo dyes are cleaved by microorganisms, forming potentially carcinogenic aromatic amines (13). This can occur in river sediments. The fragments of azo bond cleavage can undergo autoxidation under aerobic conditions, forming colored products, as in case of Acid Orange 52, where fragments form “aniline black” by polymerization (12). Azo dyes forming “forbidden aromatic amines” are not allowed to be produced in Germany (3). Among forbidden aromatic amines are some alkylated derivatives of aniline, such as 2,4,5-trimethylaniline, o-toluidine, naphthylamine derivatives, such as 2-naphthylamine, and benzidine derivatives, like aminobiphenyls. They are toxic and, as has been proven, potential carcinogens (13). It is possible that toxic metabolites are formed after hydroxylation or oxidation by cytochrome P450 (46). This creates an urgent demand for the development of multistep treatment concepts which guarantee not only irreversible decolorization but also mineralization of azo dyes. For our studies on dye degradation, we have chosen a laccase from the white rot fungus Trametes modesta which has previously shown potential for dye degradation (34). Laccases are well known as benzenediol:oxygen oxidoreductase (EC 1.10.3.2) and belong to the class of blue oxidases. Their typical molecular mass ranges from 60 to 85 kDa (10, 50). Laccases are capable of catalyzing a four-electron transfer reaction necessary to reduce molecular oxygen, which is used as the terminal electron acceptor, thus forming water without formation of H2O2. Laccases have a very broad substrate specificity with respect to the electron donor (54, 55). The enzyme catalyzes the formation of free radicals by removal of a hydrogen atom from the hydroxyl group of orthoand para-substituted mono- and polyphenolic substrates and from aromatic amines by one electron abstraction, which are capable of undergoing polymerization through radical coupling to form phenolic polymers (6, 7). Further electron withdrawal will lead to depolymerization, repolymerization, demethylation, or quinone formation (11, 19). Artificial redox mediators, such as 2,2 -azino-bis(3-ethylthiazoline-6-sulfonate, hydroxybenzotriazole, and phenothiazines, are used to extend the substrate range of laccases, e.g., to nonphenolics subunits of lignin (8) or anthracene (25). Although mediators can enhance dye degradation by laccase (8, 9, 14, 34, 52, 56), they do not have industrial potential for this process, since their application is expensive and increases wastewater toxicity. While laccases can eventually be reused by immobilization (2), mediators are lost with the effluents. As an alternative to artificial redox mediators, we have accessed the potential of ultrasound to enhance enzymatic dye degradation. Ultrasound has found a widespread industrial application, such as for surface treatment, soldering, and formation of emulsions (38, 48). Its application in organic chemistry has also become increasingly important, especially for the synthesis of organometallic compounds (29). When aqueous solutions are exposed to ultrasound, transient cavitations are formed due to compression and rarefaction of the bulk water. The collapse of 2600 * Corresponding author. Mailing address: University of Applied Sciences Cologne, Faculty of Process Engineering, Energy and Mechanical Systems, Institute of Chemical Engineering and Plant Design, Betzdorfer Strasse 2, D-50679 Cologne, Germany. Phone: 49 221 8275 2234. Fax: 49 221 8275 2202. E-mail:
[email protected]. fragmentation. 337/23 . every fourth analysis was carried out by monitoring blank activity.5. All experiments were carried out at a 100 M initial dye concentration. For liquid chromatography (LC)-mass spectrometry (MS). IP-HPLC analysis further showed that laccase treatment of Acid Orange 5 (420 nm/27 ) led to four intermediates (387/19 . 420/22 . Power input was set at P 90 W (4. IP-HPLC analysis indicated that Acid Orange 5. with one showing a high retention time compared to the parent peak. 10 liters min 1. and 417/27 ). CH3CN. Acid Orange 52 (Merck). Reactive Orange 107H. accumulation 50. hydroxyl radicals and hydrogen atoms are formed by scission of the H-O bond (48). Acid Orange 52 and Direct Blue 71 showed the highest decolorization rates (more than 50% decolorization after 2 h) at the absorption maximum in the visual region. acetate buffer 50 mM. 10 m/z. 10 mM ammonium acetate plus 10% (vol/vol) CH3CN. All other used chemicals (pro analysis [p. lyophilized solutions were dissolved in a quarter of their initial volumes. there was no significant decrease of absorption maxima in the UV region (240 to 310 nm). 5. The samples were kept frozen and were centrifuged at 10.000 K). 30 to 1. Reactive Black 5H. 40. LC (reverse-phase [RP]-HPLC). detector. 250 by 5. laccase preincubated 5 min at 0 to 100 mM NaCl was added to a 100 M solution of Direct Blue 71. 350°C. 1. 1): Acid Orange 5 (Aldrich).500 m/z. and Reactive Orange 107 (DyStar). ionization parameters. Spectra were recorded between 190 and 800 nm at a scan rate of 240 nm min 1. For the first time we report on a combined treatment of azo dyes with ultrasound and laccases. 6. while after the same time Direct Blue 71 (570 nm/26 ) showed a residual dye concentration of 10 M and the formation of several intermediates. Ion-pairing (IP)-HPLC analysis was carried out with a LaChrom System. 3. Merck HITACHI.000 V. 235/18 ).to 34-min linear gradient plus 10% min 1 B. The enzymatic treatment was performed at 40°C. Laccase production and assay.000 s. Formation of bands at 320 and 350 nm was found in the cases of Acid Orange 5 and Acid Orange 52. Their acute and chronic toxicity and carcinogenic properties render them a high pollution potential (39. PDA 800. 1 mM tetrabutylammonium hydrogen sulfate in water plus 10% (vol/vol) CH3CN. 36-min to 37-min return to 0% B. oven temperature was set at 40°C. Germany). 30%. 2005 OXIDATIVE DEGRADATION OF AZO DYES 2601 FIG. MATERIALS AND METHODS Chemicals.1 W cm 2) and P 120 W (5. Acid Orange 52. width. cavities produces locally high pressure and temperature peaks (500 bar. acetonitrile (CH3CN).000 g for 10 min to remove suspended particles prior to following analysis. UV-visible region (VIS) spectrometry was done with a Perkin-Elmer Lambda 10 spectrometer (Boston. and Direct Blue 71 were decomposed to an extent of 65 to 90% after 1 h of treatment and to an extent of 90 to 100% after 2 h of treatment. which corresponded a power uptake of 84 and 124 W and a calorimetric uptake of 24 and 50 W. Dye degradation studies. negative ESI. respectively. 4. MA). Ultrasound treatment was performed with an ultrasound device K 80 (Meinhardt Ultraschalltechnik. 125 by 4 mm) LiChroshere 60 column (Merck KgaA. B. RESULTS Treatment of azo dyes with laccase. Acid Orange 52 (444 nm/24 ) also formed four intermediates (386/20 .8 ml min 1. eluent B. respectively. RP-select B (5 M. LiChrosphere WP300. 5. which represents the main drawback of this method (15. Structures of used azo dyes which were used as sodium salt. Germany) at 850 kHz.] quality) were from Sigma. 30. Under these extreme conditions. 43). Reactive Orange 16 and Reactive Orange 107 showed no decolorization. Leipzig. The faster-eluting intermediates absorbed in the UV region (227/17 . A. 1. 3. Software Chromeleon: MS.1 Agilent.VOL. and 440/30 ). pH 4. slower-eluting intermediates . Eluent flow rate was set at 1 ml min 1 at 40°C oven temperature. For chloride inhibition studies. 71. 30°C (303K) in continuous operation mode in a stirred batch reactor. version 4.a. Direct Blue 71 (Aldrich). Gradient profile: 0 to 5 min 100% A. both acid orange dyes were nearly completely degraded. it is of outstanding interest to find a combination with another treatment to enhance dye degradation. The harvested cells were incubated 90 min at room temperature at moderate aeration. Eighty percent of these species recombine before exiting the cavitation bubble. 5. Hydrophobic and highly volatile compounds are pyrolyzed inside the cavitation bubble (24). Laccase from Trametes modesta was produced and purified as previously reported (33). Hydrophilic and volatile compounds react inside the bulk water with the ejected hydroxyl radicals and at the layer between cavitation and bulk water with supercritical water. 2. 16). final concentration (54). Enzyme activity was determined by monitoring the formation of the dimeric oxidation product from 2.5. After 2 h. The simultaneous treatment was carried out at 40°C and pH 4. followed by neutralization. All three reactive dyes were hydrolyzed at 70°C and pH 12 for 24 h. To monitor the blank activity decay. high-performance liquid chromatography (HPLC) solvents used in gradientgrade quality were from Merck. gradient as described above. UV-VIS spectroscopy showed that the azo dyes were decolorized by the enzymatic treatment at different rates.000 s. Toxicity of degradation products was measured by the respiratory inhibition of Pseudomonas putida according to a modified Deutsche Industrie Norm (DIN) standard 38412 L27 protocol. Each assay mix was preincubated 15 min at 100 liters/h air. Acid Orange 5. Eluent A. Ultrasonication of azo dyes is said to lead to nitro and nitroso aromatics (27). All reactive dyes seemed to be resistant against laccase treatment. 319/25 . DAD L-7450A (180 to 850 nm). and 5 10 9-kat/ml laccase activity. LC/MSD trap software. sheath gas. avoiding the formation of toxic degradation products.5 W cm 2) according to the manufacturer’s data. Reactions were stopped by addition of 10 mM NaF. flux rate. 5 m. Reactive Orange 16 (DyStar). 50 psi. Acid Orange 52. The letter H indicates the hydrolyzed form made from the sulfonyl ester derivative. Analysis. 34 to 36 min 90% B. Generally. Reactive Black 5 (DyStar). trap. Therefore. Reactive Orange 16H.to 30-min linear gradient plus 2% min 1 B. Direct Blue 71. 37 to 45 min 0% B. Six water-soluble azo dyes have been investigated (Fig. 0.6-dimethoxyphenol as described previously (4). 435/25 . and 555/25 . and sulfate were found as end products and oxalate and nitrite as intermediates.16 0. A chloride concentration range from 11 to 47 mM corresponding to a “real concentration” in textile wastewater (41) led to a significant reduction of the decolorization rate to 29 to 15% of its initial values (data not shown).66 0.36 0.05 0.05 0.13 2. there was an absorption increase detectable. Formate.6 M. After 1. nitrate. 25 to 80% decolorization and after 2 h. achieved. the delayed oxalate decrease was linked to a stop of the formate decrease. . 319/21 .7 0. c SE). 420/24 . 46 0. In contrast to the other reactive dyes. ENVIRON. making it impossible to detect intermediates in this region. IP-HPLC did not confirm the assumption that higher initial color concentrations cause a zero-order degradation. IP-HPLC analysis indicated that the ultrasound treatment was able to achieve complete decolorization of all investigated dyes at different degradation rates.03 0. There were no intermediates detected in the case of Reactive Orange 107 H. Both acid orange dyes and Direct Blue 71 showed high degradation rates (Table 1). 75 M. 342/23 . During sonication.69 0. Ultrasound treatment. There was no degradation detectable in the case of both Reactive Orange dyes (484 nm/18 and 444/ 18 ). Direct Blue 71 underwent complete decolorization after 23 h. 2).30 0. 228/19 .21 1. 420/22 . Acid Orange 5 was almost degraded. An acetate blank (starting concentration [c0] 200 m) showed a linear decrease from the beginning (data not shown). Reactive Orange 16 showed nearly complete decolorization after 23 h. as indicated by UV-VIS spectroscopy.11 0.43 0. 560/29 ).2 0. Generally. Formate formation was marked by a linear increase of up to 40 to 100 M. The initial oxalate formation correlates with the dye concentration. a pronounced peak occurred. and there was no correlation to the dye degradation.07d 2e 6e 4e d 1.44 0. After 1 h.24 0.36 0. 45 to 97% decolorization were c0 100 M. 354/18 . all reactive dyes showed significantly higher concentrations (9 to 43 percent). Pure oxalate solution (c0 100 M) showed rather small . 468/25 ) which were degraded after 7 h of treatment. Higher initial concentrations caused a zero-order degradation. LE UV-VIS and IP-HPLC dataa for laccase treatment Degradation rate measured by: Dye UV-VIS IP-HPLC Acid Orange 5 Acid Orange 52 Direct Blue 71 Reactive Black 5H Reactive Orange 16H Reactive Orange 107H a 0. k value.67 M. At the beginning. 1.2602 TAUBER ET AL. These top concentrations correspond to 7 to 30 percent of the theoretical concentrations. indicating a significant inhibition of the enzyme. After 80% decolorization.05 1. At 220 nm and 330 nm. acetate and oxalate underwent further degradation. The absorption maxima in the visual region decreased much faster than in the UV region. UV-VIS and IP-HPLCa data for ultrasound treatmentb Dye concn and degradation rate withc: Dye UV-VIS IP-HPLC TABLE 1. Reactive Black 5 H (567 nm/21 ) formed one intermediate by laccase treatment (515/20 ). 1. e Zero order ( M h 1).06 0.3 h of ultrasound treatment. The concentration evolution of nitrate was nearly linear from the beginning. 420/22 .5 h. MICROBIOL. Direct Blue 71 showed a pseudo-firstorder degradation rate. Eighty percent of Reactive Black 5 was decolorized after 3. the acetate formation showed linear increases and reached a maximum after 5 to 30 h (Fig.1 (6 3) 10 3 Not degraded Not degraded SE). Monocarboxylic acids (acetic and formic) have been found in traces. 573/22 . While Acid Orange 5 and 52 and Direct Blue 71 reached around 2 to 4 percent of the theoretical possible concentration. IP-HPLC analysis further showed for both acid orange dyes the formation of at least five colored degradation products (AcO5.30 (5 0) 10 2 Not degraded Not degraded Acid Orange 5 Acid Orange 52 Direct Blue 71 Reactive Black 5H Reactive Orange 16H Reactive Orange 107H a b 54 100 300 100 550 M. UV-VIS showed a very large blank absorption below 230 nm. and 350/20 . 555/28 . During ultrasound treatment of azo dyes.85 0. Complete decolorization was achieved after 9 h. It is noteworthy that the water blank showed 12 M formate after 18 h.43 k value (h 1 ).37 4. No formation of intermediates was detected for Acid Orange 52. APPL. Moreover. After 20 to 34 h. Concordant IP-HPLC and UV-VIS data illustrate good or excellent degradation of Acid Orange 5 and 52 and Direct Blue 71 by laccases and poor or no degradation of the reactive dyes. 24 M. because at least 90 percent of the dye had to be degraded until the formation of oxalate started (2 to 10 h). Additionally. A “double addition” conducted at 1 10 9 kat/ml enzymatic activity was used to proof the possible occurrence of inhibitor effects by intermediates due to the fact that laccase degradation of this dye led to a residual dye concentration. Reactive Orange 107 exhibited a moderate decolorization rate. n 3 (average absorbed in the visible region (444/27 . 561/27 . which changed to higher order after 15 min at 5 10 9 kat/ml laccase activity. the same amount of dye was added and subsequent decolorization showed the same kinetic rate as before. P 90 W. 0. Reactive Orange 16 H formed one intermediate (279/16 ). 441/26 . Direct Blue 71 and Reactive Black 5 H showed the slowest degradation (Table 2) but also a pronounced intermediate formation (373/18 . AcO52.08 0. which was followed by a concentration decrease. 430/23 . n 3 (average d First order (h 1). respectively). Carbonate nearly coeluted with nitrate and was therefore not quantifiable. ionic species (formate and oxalate) detected by ion chromatography accounted for 13 to 29 percent of the degraded carbon. The formation of intermediates also showed reproducible rates. The ion-chromatographic investigations showed good degradation of all azo dyes to ionic species. TABLE 2. The comparison between HPLC and UV-VIS data showed that formation of one peak in the UV-VIS spectra corresponds to several peaks observed by HPLC. t50 19 h) as without ultrasound action (t50 21 h).5 h).30 0. and U4)m. A higher energy input.00 Reactive Orange 16H Not degraded Reactive Orange 107H Not degraded a b 1. indicating a sulfonatobenzene structure. and identical MS/MS fragmentation compared to the occurring product peak in the solution of the treated dyes. respectively. (i) LC-MS of pure azo dyes. which can be found in case of m/z 276 (Fig. n 3 (average SE). decolorized by ultrasound ( 0.85 0. LE IP-HPLC data for laccase. It showed identical retention times. Different hydroxylation sites were confirmed by different MS/MS data. U1.a and combination treatmentsb Degradation rate with: Dye Laccase Ultrasound Combination Acid Orange 5 1. Common features in MS/MS. which was confirmed by .43 1. 318 showed the loss of 64 units. Acid Orange 52 showed 12 intermediates. 71. indicating a hydroxylation [M-H O] and one having m/z 320.73 2. the substrates were treated with laccase and ultrasound irradiation simultaneously. The initial assumption of a common laccase product of Acid Orange 5 and Acid Orange 52 was confirmed by the MS analysis of 4-(4-aminophenylazo)benzenesulfonic acid.21 1.16 0.05 h 1) and moderately TABLE 3.17 0. and 395.03 0. 76 to 120% of the theoretical concentration was reached.37 Direct Blue 71 4. 45). After 48 h. indicating a rather bad degradability. which is common for polysulfonated dyes (23. In case of simultaneous treatment.43 0. caused a higher deactivation rate (P 120 W. indicated a sulfonatobenzene structure. Ultrasound treatment of Acid Orange 5 showed five RPHPLC-separable intermediates.36 0. (ii) Structure of degradation products. 2. Interestingly. The sulfate formation showed two distinct linear increases and did not correlate with the dye degradation.26 P 90 W.44 0. LC-MS. U2. such as the loss of 80 and 64 units and m/z 156. a linear decrease was observed. and U8). U7.05 0.11 0. U3.69 0. concentration decreases during the first 10 h. the effect of a combination of these methods was investigated.18 1.05 0. MS/MS of three m/z 368 and the m/z 320 intermediates showed a loss of 80 and 64 units and an m/z 156. Since both laccase and ultrasound treatment led to efficient degradation only of selected dyes. In the combined treatment. L1).39 7. 289 showed the loss of 64 units. t50 5. A preceding 1-h ( 0. Afterwards.28).29 0. however.46) and 3-h ( 0. Direct Blue 71 showed at least two LC separated intermediates. 368. Common features in tandem mass spectrometry (MS/MS). Acid Orange 52 and Reactive Orange 16H showed small amounts of a [2 M-2H Na] and a [2 M-H] species. The reaction rates were higher than the sum of the degradation rates of laccase or ultrasound treatment alone. 2005 OXIDATIVE DEGRADATION OF AZO DYES 2603 FIG. Direct Blue 71 and Reactive Orange 107 showed the most pronounced improvements. Reaction products resulting from ultrasound treatment (P 90 W) of azo dyes as quantified by ion chromatography.VOL. This dye is scarcely decolorized by laccase (decoloration rate k 0.18 0. 3.21 0. Laccase treatment of Acid Orange 5 showed at least six RP-HPLC-separated intermediates. Direct Blue 71 and Reactive Black 5H showed [M-2H]2 and a [2 M-3H]3 species.66 0. U6. m/z 320 indicated a hydroxylation [M-H O] at different hydroxylation sites (U5.13 Acid Orange 52 2. laccase was deactivated to a similar extent (P 90 W.06 0.0 0. 409. IP-HPLC data: k value (h 1 ).04 0. The fourth one shows an m/z 186 subunit. LC-MS analysis showed for all pure azo dyes a corresponding m/z ratio [M-H] . A sequential combination treatment beginning with ultrasound followed by laccase treatment was advantageous in the case of Reactive Black 5. 3. such as the loss of 80 and 64 units and an occurring m/z 156. Laccase treatment of Acid Orange 52 resulted in at least seven intermediates.30 Reactive Black 5H 0.08 0. ultrasound. Combination of laccase and ultrasound treatment. 290.45 1. with four having an m/z 368 (Fig. identical m/z rations (m/z 276). and 366. m/z 290 (L2) was found in traces from the beginning on in the dye solution. During this treatment.44) ultrasound treatment showed the most pronounced effects during the subsequent laccase treatment. were found in the case of 276.05 0.30 0. IP-HPLC data showed (Table 3) that in all cases the simultaneous treatment gave at least the same degradation rates as the individual treatments. Our findings suggest that the highest laccase degradation rates correlate with the accessibility of the amine groups. which was hardly decolorized by laccase. Structure of LC-MS-detectable intermediates originated by laccase and ultrasound treatment of Acid Orange 5 and Acid Orange 52. This suggests that only an electron-rich phenolic ring can be oxidized by laccase (18). which was more pronounced in case of Acid Orange 52. these fragments may possess absorption maxima below the HPLC detection limit of 180 nm. Reactive Orange 16H and Reactive Orange 107H have not been investigated. MICROBIOL. and Direct Blue 71 showed the highest degradation rates and very pronounced product formation detected by UV/VIS and IP-HPLC. We have clearly shown that UV-VIS spectroscopy alone can lead to misinterpretation of the actual degradation process. FIG. ENVIRON. Double addition of Direct Blue 71 showed identical pseudofirst-order degradation rates and reproducible formation of intermediates. APPL. Reactive Black 5 was hardly decolorized. 42). and –NH2 groups because of lone electron pairs and therefrom its electron-donating character. Acid Orange 5. m/z 290 and 276 were ascribed to a sequential demethylation. At this moment the aquatic toxicity was around 10 to 15%. The lack of these intermediates in the case of Reactive Black 5H suggests on the one hand that its fragments have been degraded very fast. This observation excludes the possibility of laccase inhibition by intermediates. respectively. which may compete with the parent dye molecule for the enzyme’s catalytical centers. -OCH3. showed a small toxicity increase after 6 h. which declined to 5% after 12 h. which was reached fastest by Reactive Black 5H. A laccase from Trametes trogii preferably oxidized phenols with o-. Toxicity of dye degradation products. which increases the electronic density of the nitrogen atom. Furthermore. but still these intermediates may possess -OH. because these dyes were not decolorized by laccase treatment. 40. Ultrasound treatment revealed that generally the highest toxicity was measured at the highest concentration of intermediates. Laccase treatment of Acid Orange 5 and 52 should stop after the first attack. different MS/MS data thereof. The laccase treatment showed in the case of both acid orange dyes a significant increase of the apparent toxicity. and m/z 306 was ascribed to a demethylation and subsequent hydroxylation (U9 and U10). -NHR groups which can be attacked once again by laccase. The enzymatic degradation with fungal laccase leads to formation of quinones and hydroperoxides. slightly different scenarios occurred. In contrast to several studies on dye degradation in the literature. Direct Blue 71 was detoxified after 4 h after passing through a slight maximum. This can be explained by the N. This effect is more pronounced in cases of –OH. Reactive Black 5H. However. On the other hand. which was in contrast to . Aquatic toxicity was measured based on the respiratory inhibition of Pseudomonas putida. we have used HPLC analysis in addition to UV-VIS spectroscopy for monitoring of the reaction. -NH2 groups. A prolonged treatment—6 to 12 h—led to a detoxification. Both acid orange dyes and Reactive Orange 107H showed after 7 and 4 h. Reactive Black 5H and Reactive Orange 16H showed a good correlation between the apparent toxicity and the HPLC-detectable substances. A solution of Direct Blue 71 showed after 12 h no toxicity but contained two UV active intermediates passing through a concentration maximum.N-dimethyl group of the dye. modesta. 22. DISCUSSION In this study we have shown that acid and direct azo dyes are degraded by laccases while reactive azo dyes seemed to be resistant. p-oriented groups in ortho and/or para positions.2604 TAUBER ET AL. 3. the complete degradation of all HPLC-detectable substances. because they still posses –OH. As the ultrasound treatment proceeded. Acid Orange 52. which are not degradable (11. Acid Orange 52 was degraded twice as fast as Acid Orange 5 by the laccase from T. followed by dechlorination. and RO107H) which possess C2 groups (-SO2-C2H4-OH and –NH-CO-CH3. RO16H. Galindo et al. The final nitrate concentration is much higher than the theoretical. Vinodgopal et al. acetate is degraded to succinic.N-dimethyl group of Acid Orange 52 (m/z 277) take place. Joseph et al. 2005 OXIDATIVE DEGRADATION OF AZO DYES 2605 results previously found (1.0 h 1 at 500 kHz.4 to 3. the formation of CO2. [M-H 2O] ) before their decolorization. respectively). In the case of Acid Orange 52. which was not further degraded. LC-MS proved a common product of these dyes to be most probably 4-(4-aminophenylazo)-benzenesulfonic acid. Hydroxylation occurs at different sites of the target molecule. hydroquinone. Ipso attacks on the N. Our experiments showed that Acid Orange 52 was demethylated. and acetic acid as final products. hardly scavenges hydroxyl radicals (31). 288 K. including the attack of the azo link bearing carbon. Sulfate. This dye undergoes tautomerization to a ketohydrazine derivative (37). 34). Sulfate was formed through the attack on –SO3H and –SO2groups. have also been postulated (37).7 h 1 (c0 100 M). which possibly recombines and disproportionates to an oxalate ion. If one of these groups is more easily accessible by ˙OH attack. The enrichment of low-molecular-weight species. maleic. The experimental data do not support this assumption. They observed dicarboxylic. possibly formate was formed through the primary ˙OH attack on the C1 and C2 groups. which were more pronounced in case of Acid Orange 52. formic. Our work shows that the aquatic toxicity is not clearly linked to the concentration of detected intermediates. seems to be responsible for that observation. and CH4 depends on the initial acetate concentration. hence leading to worse accessibility of the amino group by sterical hindrance. which can bind on the T2/T3 trinuclear copper cluster site inhibiting the internal electron transfer. Due to the laccase treatment. and para-benzoquinone as primary products. 53). carboxylic acids. The formation of significant concentrations of carbon-containing substances (formate and oxalate) in the case of pure water may be explained by the dissolved amount of CO2. a laccase from Rhizoctonia praticola showed detoxification of cresols and chlorophenol compounds by cross-linking them with naturally occurring phenols (7). Possibly. There is a . the pseudofirst-order degradation changes to higher orders independently of the initial dye concentration. Possibly aromatic amines are formed. it is difficult to predict the further degradation pathways of these intermediates. In contrast. Various textile dyes have been decolorized by a Trametes hirsuta laccase to an extent of 80% and showed no general rule in detoxification tendencies (2).4-dichlorophenoxyacetic acid is first exposed to an ipso attack of OH radicals on the ether substituent. Generally. which was not confirmed by IP-HPLC. because their fate depends on their physical and chemical properties. there has been found 50% residual total organic carbon concentration (27). which is also possible for Reactive Orange 16. which confirms what has been stated above. 186). both acid orange dyes exhibited significant toxicity increases during the first 6 h. The concentration profile indicated that oxalate was an intermediate which was possibly degraded to formate. Pure oxalate was hardly degraded during the first 10 h. During the first 10 h of azo dye degradation. the degree of mineralization to gaseous compounds strongly depends on the on the gas mixture (5. or in the “bulk” water. It is well known that carbon dioxide is in equilibrium with carbonate and hydrocarbonate (32). 50 W. Peller et al. Ultrasound degradation of Acid Orange 52 and Direct Blue 71 followed by UV-VIS showed a zero-order decolorization rate at a high initial dye concentration. 71. Acid Orange 52 was degraded at 1. c0 10 M) (27). (27) assumed that the ˙OH attack on the same dye leads to hydroxyl amines (which were not detected by MS analysis). Further reactions may occur inside the cavitation. the flexion point in the concentration profile should lie on a stoichiometric point. Later-occurring formate may be formed by acetate and oxalate degradation. (49) described the attack of nonvolatile Reactive Black 5 in the “bulk” water by ˙OH radicals which destroy the chromophoric system through azo bond cleavage. As the reaction proceeds. Phenol degradation led to catechol. Both acid orange dyes and their intermediates—except one—are degraded completely. no quantification of nitrate originating from dyes was possible. The late oxalate formation indicated its origin in primary intermediates and not in the azo dyes themselves. oxalate was attacked by the accumulating hydrogen peroxide. which indicated that these C2 groups are essential for acetate formation. in the hypercritical water layer. The highest acetate concentrations were reached by reactive dyes (RBl5H. The enzyme laccase generally is very sensitive to halide ions and small anions (18. which is in good agreement with results found by other investigators (2. propionic. (17) found that Acid Orange 52 was demethylated by UV-H2O2 treatment—a treatment which produced OH radicals—and carboxylic acids and aliphatic compounds have been formed as final products. Independent investigations reported in the literature have shown that total organic carbon decreases until a residual concentration is left around 20% for Reactive Black 5. These observations may be explained by the broad substrate specificity of laccase or the possible action of internal mediators. Further. succinic. because the N2 oxidation originated from air-saturated solutions (48). Different mechanisms.VOL. followed by subsequent oxidation. the aquatic toxicity slightly increased. The latter is transformed by ˙OH attack to a carbonate radical (17). and acetic acid. The degradation rate of Direct Blue 71 was reduced to 29 to 15% of its initial values by chloride concentrations commonly found in textile wastewater. which was confirmed by LC-MS/MS results (m/z 156. (35) found out that 2. As a matter of fact. leading to nitroso and nitro aromatic compounds. which are intermediates formed after the first laccase attack. CO. a product of ultrasound treatment. since they are more difficult to degrade and may act as quencher (47). (21) have found that under argon saturation. LC-MS proved monohydroxylation and bihydroxylation of the azo dyes ([M-H] 3 [M-H O] . which was ascribed to oxalate (49). such as oxalic. and glycolic acids. 36). The two main intermediates of Direct Blue 71 are possibly products. Therefore. Acetate intermediate was most probably degraded to formate. Although Reactive Black 5H was degraded very slowly. like oxalic acid. After 2 h. Gutierrez et al. the apparent toxicity reached its highest value and declined afterwards very fast to 0%. leading to phenyl derivative radicals. were identified as final products (5). In addition to CO2. glyoxylic. and B. A. L. J. and D. 70: 409–414. and V. and P. Erlacher (TU Graz). R. J.. E. and S. I. C. E. Steiner. D. 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