Basic and applied aspects in the microbial degradation of azo dyes

Appl Microbiol Biotechnol (2001) 56:69–80 DOI 10.1007/s002530100686 MINI-REVIEW A. Stolz Basic and applied aspects in the microbial degradation of azo dyes Received: 23 February 2001 / Received revision: 5 April 2001 / Accepted: 6 April 2001 / Published online: 6 June 2001 © Springer-Verlag 2001 Abstract Azo dyes are the most important group of synthetic colorants. They are generally considered as xenobiotic compounds that are very recalcitrant against biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several microorganisms are able, under certain environmental conditions, to transform azo dyes to non-colored products or even to completely mineralize them. Thus, various lignolytic fungi were shown to decolorize azo dyes using ligninases, manganese peroxidases or laccases. For some model dyes, the degradative pathways have been investigated and a true mineralization to carbon dioxide has been shown. The bacterial metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond, which results in the formation of (usually colorless) amines. These reductive processes have been described for some aerobic bacteria, which can grow with (rather simple) azo compounds. These specifically adapted microorganisms synthesize true azoreductases, which reductively cleave the azo group in the presence of molecular oxygen. Much more common is the reductive cleavage of azo dyes under anaerobic conditions. These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the organisms involved and the dyes converted. In these unspecific anaerobic processes, low-molecular weight redox mediators (e.g. flavins or quinones) which are enzymatically reduced by the cells (or chemically by bulk reductants in the environment) are very often involved. These reduced mediator compounds reduce the azo group in a purely chemical reaction. The (sulfonated) amines that are formed in the course of these reactions may be degraded aerobically. Therefore, several (laboratory-scale) continuous anaerobic/aerobic processes for the treatment of wastewaters containing azo dyes have recently been described. A. Stolz (✉) Institut für Mikrobiologie der Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany e-mail: Andreas.Stolz@PO.Uni-Stuttgart.de Tel.: +49-711-6855489, Fax: +49-711-6855725 Introduction Azo dyes are characterized by the presence of one or more azo groups (-N=N-). They are the largest and most versatile class of dyes, and more than half of the annually produced amount of dyes (estimated for 1994 worldwide as 1 million tons) are azo dyes. Presumably more than 2,000 different azo dyes are currently used to dye various materials such as textiles, leather, plastics, cosmetics, and food. The largest amount of azo dyes is used for the dyeing of textiles, and it had been estimated that about 10% of the dye-stuff used during these dyeing processes does not bind to the fibers and is therefore released into sewage treatment systems or the environment (Anliker 1979; Chudgar 1985; Clarke and Anliker 1980; Reisch 1996; Zollinger 1991). In particular, the soluble reactive dyes, which are being used in increasing quantities, are known to hydrolyze during application without a complete fixation, which may result in an even larger proportion of these dyes being released into the environment (Carliell et al. 1994; Jeckel 1997; Weber and Stickney 1993). There is only a single example for the presence of an azo group in a natural product (4,4′-dihydroxyazobenzene; Gill and Strauch 1984) and the industrially produced azo dyes are therefore all xenobiotic compounds. It is thus not surprising that azo dyes usually resist biodegradation in conventional aerobic sewage-treatment plants (Pagga and Brown 1986; Shaul et al. 1991). The recalcitrance of the azo dyes to biological degradative processes results in severe contamination of the rivers and ground water in those areas of the world with a high concentration of dyeing industries (Maguire and Tkacz 1991; Namasivavayam and Yamuna 1992; Ràfols and Barceló 1997; Riu et al. 1998; Tincher and Robertson 1982). The current state of the art for the treatment of wastewaters containing dyes are physicochemical techniques, such as adsorption, precipitation, chemical oxidation, photodegradation, or membrane filtration (e.g. Churchley 1994; Panswed and Wongehaisuwan 1986; Yeh and 70 Thomas 1995; Yoshida et al. 1991). All of these have serious restrictions as economically feasible methods for decolorizing textile wastewaters (such as high cost, formation of hazardous by-products or intensive energy requirements). This has resulted in considerable interest in the use of biological systems for the treatment of these wastewaters. In the present review the fundamental biological reactions that allow the transformation of azo dyes are discussed and a short survey of possible technical applications of these reactions to the treatment of wastewaters from the textile industry is given. Aerobic decolorization of azo dyes by lignin-degrading fungi The first report of aerobic degradation of azo dyes by lignolytic fungi appeared in 1990, when Cripps et al. demonstrated that nitrogen-limited cultures of Phanerochaete chrysosporium decolorized the azo dyes Acid Orange 7 (Orange II), Acid Orange 6 (Tropaeolin O), or Direct Red 28 (Congo Red) (Fig. 1 A–C). Subsequent work demonstrated that cultures of P. chrysosporium also decolorized several other azo dyes (Banat et al. 1996; Young and Yu 1997). Currently, there is no correlation known between the structure of the azo dyes and the ability of P. chrysosporium to degrade the dyes (Pasti-Grigsby et al. 1992; Paszczynski et al. 1992). Experiments using 14C-labeled azo dyes demonstrated that simple non-sulfonated azo dyes (e.g. Disperse Yellow 3; Fig. 1D) and also sulfonated dyes containing radiolabeled sulfanilic acid (4-aminobenzenesulfonic acid) as structural elements (e.g. Acid Orange 7, Fig. 1A, or Acid Yellow 9, Fig. 1E) were degraded by P. chrysosporium to 14CO (Paszczynski et al. 1992; Spadaro et al. 1992). 2 More recently, it has been shown that not only P. chrysosporium but also several other fungi (mainly white rot fungi) (e.g. Geotrichum candidum, Trametes versicolor, Bjerkandera adusta, Penicillium sp., Pleurotus ostreatus, Pycnoporus cinnabarinus, and Pyricularia oryzae) are able to decolorize rather complex azo dyes, such as Direct Blue 1 (Chicago Sky Blue 6B) (Fig. 1F) or the reactive dye Reactive Black 5 (Fig. 1G). Recent comparisons of different fungi suggested that other fungi (e.g. Trametes or Bjerkandera species) are superior compared to P. chrysosporium for the decoloration of different dyes (Chivukula and Renganathan 1995; Heinfling et al. 1997; Kim et al. 1995; Knapp et al. 1995; Rodríguez et al. 1999; Schliephake et al. 2000; Shin and Kim 1998; Swamy and Ramsay 1999a; Zheng et al. 1999). Function of lignin and manganese peroxidases and laccases in the fungal degradation of azo dyes Fig. 1A–I Examples of azo compounds that are decolorized by (lignolytic) fungi. A Acid Orange 7 (Orange II); B Acid Orange 6 (Tropaeolin O); C Direct Red 28 (Congo Red); D Disperse Yellow 3; E Acid Yellow 9; F Direct Blue 1 (Chicago Sky Blue 6B); G Reactive Black 5; H Acid Red 66 (Biebrich Scarlet); I Acid Yellow 23 (Tartrazine) (Cripps et al. 1990; Heinfling et al. 1998; Paszczynski and Crawford 1992; Paszczynski et al. 1992; Schliephake et al. 2000; Spadaro et al. 1992) The ability of P. chrysosporium and other fungi to degrade azo dyes is generally correlated with the ability of these organisms to synthesize lignin-degrading exoenzymes such as lignin- and manganese peroxidases or laccases (Chivukula and Renganathan 1995; Heinfling et al. 1998; Kim and Shoda 1999; Schliephake et al. 2000). Lignin and manganese peroxidases show a similar reaction mechanism and are oxidized during their catalytic cycle by H2O2 to an oxidized state which is reduced by the substrates (e.g. azo dyes) in two subsequent oneelectron transfer steps to the native form of the enzyme. While lignin peroxidases are able to oxidize nonphenolic aromatic compounds, manganese peroxidases preferentially oxidize Mn2+ to Mn3+, and the Mn3+ is responsible for the oxidation of many phenolic compounds. Laccases are copper-containing enzymes produced by a number of plants and fungi which oxidize phenols and anilines in the presence of oxygen (Barr and Aust 1994; Glenn et al. 1986; Thurston 1994). It was shown for P. chrysosporium that lignin peroxidase and manganese peroxidase (in the presence of Mn2+) were both able to decolorize azo dyes and that both enzymes showed differences in substrate specificity towards different azo dyes (Pasti-Grigsby et al 1992; 71 Paszczynski et al. 1991). The activity of the lignin peroxidase from P. chrysosporium with certain azo dyes [such as Acid Red 66 (Biebrich Scarlet), Fig. 1H, and Acid Yellow 23 (Tartrazine), Fig. 1I] was significantly enhanced by the addition of the mediator compound veratryl alcohol. Similar increases in the reaction rates have also been observed for the oxidation of other organic substrates by this enzyme (Bumpus 1995; Ollikka et al. 1993; Paszczynski and Crawford 1991). It was originally assumed that manganese peroxidases and laccases would only convert a rather limited spectrum of azo dyes and preferentially convert dyes which carry a phenolic substituent in para-position to the azo bond and additional methyl- or methoxy-substituents in 2- or 2,6-position in relation to the hydroxy-group (Chivukula and Renganathan 1995; Pasti-Grigsby et al. 1992). More recently it was shown that certain manganese peroxidases (e.g. from Bjerkandera adusta) or laccases (e.g. from Pycnoporus cinnabarinus) are also able to decolorize complex industrially relevant azo dyes, such as Reactive Black 5 (Fig. 1G) or Direct Blue 1 (Fig. 1F) (Heinfling et al. 1998; Schliephake et al. 2000). Elucidation of the degradative pathways utilized by white rot fungi for the decoloration of azo dyes The oxidation of the non-sulfonated azo dye 1-(4′-acetamidophenylazo)-2-naphthol (a structural analogue of the industrially relevant azo dye Disperse Yellow 3) by the lignin peroxidase from P. chrysosporium resulted in the formation of 1,2-naphtoquinone and acetanilide (Fig. 2). This suggested that the oxidized form of the lignin peroxidase abstracted two electrons from the phenolic ring of the dye, which resulted in formation of the corresponding carbonium ion on the C-1 carbon of the naphthol ring. This carbonium ion can then be hydrated by a nucleophilic attack of water to an intermediate that breaks down to the naphthoquinone and an unstable phenyldiazene. It was suggested that this phenyldiazene could be oxidized by molecular oxygen to the corresponding radical, which finally splits off molecular nitrogen under formation of the phenyl radical, which is stabilized by the abstraction of a hydrogen radical from its surroundings (Spadaro and Renganathan 1994). The enzymatic mechanism for the oxidation of sulfonated azo dyes by fungal peroxidases has been studied independently by two different groups who presented slightly different results. Goszczynski et al. (1994) incubated 3,5-dimethyl-4-hydroxyazobenzene-4′-sulfonic acid and 3-methoxy-4-hydroxyazobenzene-4′-sulfonamide with a crude peroxidase preparation from P. chrysosporium and analyzed the products formed using mass spectroscopy. From the identified products, they also suggested an initial oxidative activation of the dyes with the formation of a carbonium ion followed by a nucleophilic attack of water on this cationic species. From the metabolites observed, it was suggested that this unstable tetrahedral intermediate could either break down by a sym- Fig. 2 Proposed reaction mechanisms for the oxidation of 1-(4′acetamidophenylazo)-2-naphthol (left) and 3,5-dimethyl-4-hydroxyazobenzene-4′-sulfonate (right) by the lignin peroxidase from Phanerochaete chrysosporium (Chivukula et al. 1995; Goszczynski et al. 1994; Spadaro and Renganathan 1994) metric cleavage of the azo group (which produces a quinone imine and a nitroso compound) or an asymmetric cleavage (resulting in a quinone and a phenyldiazene). These direct oxidation products should finally undergo various spontaneous reactions that finally result in the formation of various secondary products. In the second study, a purified lignin peroxidase preparation from P. chrysosporium was used for the oxidation of 3,5-dimethyl-4-hydroxyazobenzene-4′-sulfonic acid and Acid Orange 7 (Chivukula et al. 1995). In contrast to the previous study, a 4-sulfophenylhydroperoxide was found as major product formed from 3,5-dimethyl4-hydroxyazobenzene-4′-sulfonic acid and Acid Orange 7. The second aromatic system of the azo dyes was also converted according to these authors to the corresponding quinones. It was suggested that the differences in the products formed from the non-sulfonated and the sulfonated azo dyes were due to differences in the reactivity between phenyl radicals and sulfophenyl radicals (Chivukula et al. 1995). The formation of the same products (2,6-dimethoxybenzoquinone and 4-sulfophenylhydroperoxide) was also described for the oxidation of 3,5-dimethyl-4-hydroxyazobenzene-4′-sulfonic acid by a laccase from Pyricularia oryzae (Chivukula and Renganathan 1995). 72 Degradation of azo dyes by bacterial peroxidases In the course of investigating the degradation of azo dyes by lignolytic fungi, it was discovered that also some peroxidase-producing bacterial strains (mainly Streptomyces species, but also gram-negative bacteria such as Sphingomonas chlorophenolicus=“Flavobacterium ATCC 39723”) decolorize azo dyes (Cao et al. 1993; Paszczynski et al. 1992). The oxidation of azo dyes by Streptomyces chromofuscus A11 involved an extracellular peroxidase that showed a restricted substrate specificity similar to that of the manganese peroxidase from P. chrysosporium or horseradish peroxidase (Pasti-Grigsby et al. 1992, 1996). In contrast to the lignolytic fungi, the peroxidaseproducing bacteria studied produced only insignificant amounts of 14CO2 from industrially relevant 14C-labeled azo dyes (Paszczynski et al. 1992). Cometabolic reductive cleavage of azo dyes by aerobic bacteria During the last years, several bacterial strains have been described that aerobically decolorize azo dyes by reductive mechanisms (for an overview of these the organisms, see Banat et al. 1996). Many of these isolates decolorize the azo compounds only in the presence of other carbon sources and therefore presumably do not use the azo dyes as carbon or energy sources. Thus a Bacillus subtilis strain was studied that reductively cleaved paminoazobenzene (Fig. 3A) to aniline (and presumably p-phenylendiamine) during aerobic growth on glucose (Zissi et al. 1997). Similarly, strains of Pseudomonas stutzeri, Acetobacter liquefaciens, and Klebsiella pneumoniae were able to reductively cleave 4′-dimethylaminoazobenzene-2-carboxylic acid [Acid Red 2 (Methyl Red), Fig. 3B] during aerobic growth on Nutrient Broth or glucose (Wong and Yuen 1996; Yatome et al. 1993). Furthermore, the reductive decolorization of sulfonated azo dyes (e.g. Acid Orange 7, Fig. 1A, Acid Orange 10, Acid Red 88, Acid Red 4, Acid Orange 8 Fig. 3C–F) by different bacterial strains (Bacillus sp., Pseudomonas sp., Sphingomonas sp., Xanthomonas sp.) under aerobic conditions in the presence of additional carbon sources has been reported (Coughlin et al. 1997, 1999; Dykes et al. 1994; Jiang and Bishop 1994; Sugiura et al. 1999). In many reports on the “aerobic” metabolism of azo dyes, the bacterial strains (e.g. Aeromonas sp., Bacillus subtilis, Proteus mirabilis, Pseudomonas pseudomallei 13NA, Pseudomonas luteola) were grown aerobically with complex media or sugars and then incubated (often using high cell densities) without shaking in the presence of different azo dyes (Chang and Lin 2000; Chen et al. 1999; Hayase et al. 2000; Horitsu et al. 1977; Idaka et al. 1978; Ogawa et al. 1986; Yatome et al. 1981). These resting cell cultures presumably become rapidly oxygendepleted, and the reactions observed should therefore be viewed as an anaerobic incubation of azo dyes (see below). Fig. 3A–J Examples of azo compounds that are decolorized by aerobic bacteria. A p-Aminoazobenzene; B 4′-dimethylaminoazobenzene-2-carboxylic acid (Methyl Red); C Acid Orange 10; D Acid Red 88; E Acid Red 4; F Acid Orange 8; G 4,4′-dicarboxyazobenzene; H 4-carboxy-4′-sulfoazobenzene; I 1-(4′-carboxyphenylazo)-4-naphthol (“carboxy-Orange I”), J 1-(4′-carboxyphenylazo)-2-naphthol (“carboxy-Orange II”) (Blümel et al. 1998; Coughlin et al. 1999; Kulla 1981; Overney, 1979; Yatome et al. 1993; Zissi et al. 1997) Aerobic growth of bacteria with azo dyes as sole source of carbon and energy There are several claims in the literature that bacteria with the ability to reduce azo dyes aerobically in a cometabolic fashion can also use these dyes as sole source of carbon and energy (e.g. Dykes et al. 1994; Yatome et al. 1993); however, there are very few studies that unequivocally demonstrate the utilization of azo compounds as sole source of carbon and energy under aerobic conditions. The ability of bacteria to grow with simple carboxylated azo compounds as sole source of carbon and energy was first shown by Overney (1979), who isolated a "Flavobacterium" that was able to grow aerobically with the simple model compound 4,4′-dicarboxyazobenzene (Fig. 3G). In a later study it was demonstrated that after enrichments with 4,4′-dicarboxyazobenzene a wide range of bacterial strains could be readily isolated from different inocula. These strains were classified according to the Biolog test system and found to belong to different genera, such as Sphingomonas, Comamonas, Pseudomonas, Xanthomonas, or Alcaligenes (Hausser 1995). In a now almost classical study on the potential of bacteria to acquire novel metabolic traits, Kulla, Leising- 73 er and coworkers demonstrated that a mixed bacterial culture which degraded 4,4′-dicarboxyazobenzene could be adapted to the degradation of more complex azo compounds such as 1-(4′-carboxyphenylazo)-4-naphthol (“carboxy-Orange I”) (Fig. 3I) or 1-(4′-carboxyphenylazo)-2-naphthol (“carboxy-Orange II”) (Fig. 3J). From these adaptation processes in continuous cultures, strain “Pseudomonas” K22 was obtained after cultivation with “carboxy Orange I” and strain KF46 from an enrichment with “carboxy Orange II” (Kulla 1981; Kulla et al. 1984). A recent taxonomic study, which was performed with two direct descendants of these strains, which are currently still available (strain K24 and strain KF46F), demonstrated that both strains belong to two new genera in different families within the β-subgroup of the Proteobacteria. Thus strain K24 was described as a member of the Alcaligenaceae (Pigmentiphaga kullae) and strain KF46F as a member of the Comamonadaceae (Xenophilus azovorans) (Blümel et al. 2001a, b). The subsequent attempts of Kulla and coworkers to adapt the “carboxy-Orange”-degrading bacterial strains K22 and KF46 to grow with the structurally analogous sulfonated dyes Acid Orange 20 (Orange I) and Acid Orange 7 (Orange II) were not successful, and it was suggested that the intermediate formation of 4-aminobenzenesulfonate (sulfanilate) somehow interfered with the central metabolism of the bacteria (Kulla et al. 1983). Therefore it was later attempted to adapt the sulfanilatedegrading strain Hydrogenophaga palleronii strain S1 (recently reclassified as H. intermedia, Contzen et al. 2000) to grow with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (Fig. 3H) as sole source of carbon and energy. This resulted finally in the isolation of a mutant strain of strain S1 (called strain S5) that grew with the simple sulfonated azo dye as sole source of carbon and energy. Strain S5 metabolized 4-carboxy-4′sulfoazobenzene reductively to 4-aminobenzoate and sulfanilate, which were mineralized by previously established degradative pathways (Blümel et al. 1998; Feigel and Knackmuss 1993). Recently, evidence has been presented that Sphingomonas 1CX, which cometabolically decolorized several sulfonated azo dyes (see above), also grew with (low concentrations) of Acid Orange 7 (Coughlin et al. 1999). The aerobic azoreductases During the aerobic, “semi-aerobic” (in static culture) or anaerobic incubation of bacteria with azo compounds, amines were often detected that originated from a reductive cleavage of the azo bond. The aerobic reductive metabolism of azo dyes requires specific enzymes (“aerobic azoreductases”) that catalyze these reactions in the presence of molecular oxygen. The aerobic azoreductases from the “carboxy-Orange”-degrading strains K22 and KF46 were purified, characterized and compared with each other (Zimmermann et al. 1982, 1984). Both azoreductases were monomeric flavin-free enzymes that pref- erentially used NADPH (and only with significantly higher Km values NADH) as cofactors and reductively cleaved not only the carboxylated growth substrates of the bacteria but also the sulfonated structural analogues. Both enzymes significantly differed in size (21 kDa vs 30 kDa) and substrate specificity. The azoreductase from strain KF46 (Orange II azoreductase) strictly required the presence of a hydroxy-group in ortho-position to the azo bond. In contrast, the Orange I azoreductase from strain K22 required a hydroxy-group in para-position to the azo bond for catalytic activity. Surprisingly, neither of the purified enzymes exhibited immunological crossreaction with each other, which suggests that the two enzymes are evolutionary significantly different (Zimmermann et al. 1982, 1984). More recently, the purification and characterization of enzymes from Shigella dysenteriae and Escherichia coli with flavin-dependent aerobic azoreductase activities has been reported (Ghosh et al. 1992, 1993). Unfortunately, the authors used assay conditions that did not allow a clear distinction between a true aerobic azoreductase (the existence of which would be very surprising in these well-characterized bacterial species, given the long search by different groups for aerobic azoreductases) and a reaction caused by the intermediate formation of reduced flavins by flavin reductase activities, which then could unspecifically reduce the azo dyes (see below) (Russ et al. 2000). Anaerobic reduction of azo dyes by bacteria In contrast to the few reports of aerobic decolorization of azo dyes, a wide range of organisms are able to reduce azo compounds under anaerobic conditions. This has been shown for purely anaerobic (e.g. Bacteroides sp., Eubacterium sp; Clostridium sp.), facultatively anaerobic (e.g. Proteus vulgaris, Streptococcus faecalis), and aerobic (e.g. Bacillus sp., Sphingomonas sp.) bacteria, yeasts, and even tissues from higher organisms (Adamson et al. 1965; Bragger et al. 1997; Dieckhues 1960; Dubin and Wright 1975; Mecke and Schmähl 1957; Rafii et al. 1990; Scheline et al. 1970; Walker 1970; Wuhrmann et al. 1980). The main interest in this field has been focused on bacteria from the human intestine that are involved in the metabolism of azo dyes ingested as food additives (Chung et al. 1992). The unspecificity of this reaction is also demonstrated by the many reports of decolorization of azo dyes by anaerobically incubated sewage sludge (e.g. Bromley-Challenor et al. 2000; Brown and Laboureur; 1983a; Carliell et al. 1994; Delée et al. 1998; Ganesh et al. 1994). It appears that almost every azo compound that has been tested is biologically reduced under anaerobic conditions, although there are some indications that metal-ion-containing dyes sometimes have reduced decolorization rates (for a survey of compounds tested see Brown and DeVito 1993; Chung et al. 1978, 1992; Delee et al. 1998; Diekhues 1960). 74 Fig. 4 Proposed mechanism for the redox-mediator-dependent reduction of azo dyes by Sphingomonas xenophaga BN6. AR Azoreductase, RM redox mediator (Keck et al. 1997) Mechanisms for the unspecific reduction of azo dyes under anaerobic conditions The physiology of the possible reactions that result in a reductive cleavage of azo compounds under anaerobic conditions differs significantly from the situation in the presence of oxygen, because several redox active compounds (e.g. reduced flavins or hydroquinones) rapidly react either with oxygen or with azo dyes. Therefore, under aerobic conditions oxygen and the azo compounds compete for the reduced electron carriers. The spontaneous reactions of the reduced forms of these electron carriers (or mediator compounds) with the azo dyes allows for very unspecific reduction processes, which are mainly governed by the redox potentials of the redox mediators and the azo compounds. In earlier studies with facultatively anaerobic bacteria, it was repeatedly suggested that reduced flavins generated by cytosolic flavin-dependent reductases (flavin reductases) were responsible for the unspecific reduction of azo dyes (Roxon et al. 1967; Walker 1970). The ability of cytosolic flavin reductases to act in vitro as azoreductases was recently demonstrated by experiments using a recombinant flavin reductase in different genetic backgrounds (Russ et al. 2000). Also, the addition of other putative redox mediators (e.g. benzyl viologen or quinones) to cultures of strictly anaerobic bacteria significantly increased the reduction rate of azo dyes (Bragger et al. 1997; Brown 1981; Chung et al. 1978). Cell extracts show generally much higher rates for the anaerobic reduction of azo dyes than do preparations of resting cells (Mechsner and Wuhrmann 1982; Walker 1970; Wuhrmann et al. 1980). This has generally been explained by the low permeability of the cell membranes for the highly polar sulfonated azo compounds. Therefore, it appears reasonable that, in vivo, intracellular enzymes like flavin reductases are of little importance for the reduction of sulfonated azo compounds (Russ et al. 2000). A different model for the unspecific reduction of azo dyes by bacteria which does not require transport of the azo dyes or reduced flavins through the cell membranes was recently suggested for Sphingomonas xenophaga BN6. The anaerobic reduction of azo compounds by this strain was significantly increased after the addition of different quinones, such as anthraquinone-2-sulfonate or 2-hydroxy-1,4-naphthoquinone. It was suggested that in this system the quinones acted as redox mediators which were enzymatically reduced by the cells of S. xenophaga BN6 and that the hydroquinones formed reduced the azo dyes in the culture supernatants in a purely chemical redox reaction (Fig. 4). Cell fractioning experiments demonstrated that the quinone reductase activity was located in the cell membranes of S. xenophaga BN6 and that therefore no transport of the sulfonated azo compounds or of the hydroquinone/quinone redox mediator via the cell membrane was necessary (Kudlich et al. 1997). Furthermore, it was demonstrated that (probably quinoide) redox mediators, active in the reduction of azo dyes, were also formed by S. xenophaga BN6 during growth with naphthalenesulfonates (Keck et al. 1997). The involvement of membrane-bound enzyme systems (e.g. NAD(P)H-cytochrome c reductase or the cytochrome P450 system) in the anaerobic reduction of azo dyes has also been described for mammalian cells (Brown and deVito 1993; Hernandez et al. 1967a, 1967b; Zbaida 1995). Yet another model for the reduction of sulfonated azo compounds, one which also does not require membrane transport of the dyes, has been suggested for certain strictly anaerobic bacterial strains from the intestine. Rafii and coworkers isolated different bacteria from the human intestine (e.g. Eubacterium sp., Clostridium sp., Butyrvibrio sp., or Bacteroides sp.) that decolorized sulfonated azo dyes during growth on solid or liquid complex media. It was shown that at least part of the azoreductase activities were extracellular, because the culture supernatants were able to decolorize the dyes under anaerobic conditions (Rafii et al. 1990, 1995). The azoreductase activity from Clostridium perfringens was described as being independent of added flavins; furthermore, the enzyme was rapidly and irreversibly inactivated by oxygen (Rafii et al. 1990). It is still unclear in this system how the supposed extracellular azoreductases gain the NADH necessary for the reduction of the azo dyes in their extracellular environment and if there are some effects that are caused by the complex growth media of the cells. Another possibility for the extracellular reduction of azo compounds by microorganisms is the action of reduced inorganic compounds (e.g. Fe2+, H2S) that are formed as end-products of certain strictly anaerobic bacterial metabolic reactions on the azo bond. Thus it has been recently shown that the formation of H2S by sulfate-reducing bacteria resulted in the reduction of the azo dye Reactive Orange 96 (Libra et al. 1997; Yoo et al. 1999). In the environment, presumably also “bulk reductants” such as Fe2+ or H2S will show significantly increased reaction rates in the presence of mediator compounds (Schwarzenbach et al. 1990; Perlinger et al. 1996). In summary, it appears that under anaerobic conditions in the environment or in sewage treatment systems, specific azoreductases (if they exist at all) are probably only of limited importance for the reduction of azo dyes. This is in sharp contrast to the requirement for true azo- 75 reductases under aerobic conditions and readily explains the ubiquitous spread of the ability of microorganisms to reduce azo compounds under anaerobic conditions. Possible applications of microorganisms for the treatment of dye-containing waste waters It is generally observed that in conventional aerobic sewage-treatment plants most azo dyes are not degraded by the bacteria, but that a certain percentage (usually about 40–80%) of the dyes physically adsorb to the sewage sludge (Clarke and Anliker 1980; Dohányos et al. 1978; Hitz et al. 1978; Shaul et al. 1991; Pagga and Brown 1986; Pagga and Taeger 1994; Shaul et al. 1991). This correlates well with the observed difficulties when the isolation of bacteria with “aerobic azoreductase” activity is attempted (see above). Therefore, conventional aerobic sewage-treatment systems are not useful for the decolorization of effluents containing azo dyes and various advanced chemo-physical techniques are necessary for the treatment of textile wastewater (Schönberger 1997). In the textile processing industry, a wide range of structurally diverse dyes is used within short time periods in one and the same factory, and therefore effluents from the textile industry are extremely variable in composition (Correia et al. 1995). This underlines the need for a largely unspecific process for the treatment of textile wastewater. From the currently known biological systems, the required unspecifity may be obtained by using either the lignin peroxidases from lignolytic fungi or the unspecific reduction processes catalyzed by various bacteria under anaerobic conditions. Although Zhang et al. (1999) recently demonstrated that a white rot fungus was able to stably decolorize Acid Orange 7 (Orange II) in a bioreactor for 2 months, it appears that currently there are severe problems which interfere with the utilization of lignolytic fungi for the treatment of dye-containing wastewaters: ● ● ● Wastewater treatment plants are not the natural habitat of lignolytic fungi and therefore special care has to be taken to establish these fungi in a wastewater treatment system. The lignolytic enzymes of the white rot fungi are thought to be expressed in most cases only during secondary metabolism following growth when carbon and/or nitrogen sources become limiting. Neither lignin nor any of the pollutants degraded by the enzymes has been shown to be utilized as a carbon or energy source, and a separate carbon source is required for the cultivation of the organisms (Swamy and Ramsay 1999b). The observed degradation rates are usually rather low. In typical experiments about 50–150 mg of the respective dyes/l are decolorized within 5–10 days (Chao and Lee 1994; Pasti-Grigsby et al. 1992; Paszczynski et al. 1992; Hardin et al. 2000; Swamy and Ramsay 1999a, b). ● ● Lignin peroxidases are very unspecific for the oxidation of aromatic and xenobiotic compounds. Therefore, in the presence of complex substrate mixtures such as those observed in industrial sewage-treatment systems, also other substrates will be oxidized by lignin peroxidases. Lignin peroxidases exhibit a pH-optimum at pH 4.5–5. Therefore a rather acid pH of the wastewater treatment system is required, which may inhibit the growth of several other useful microorganisms (Swamy and Ramsay 1999a). Based on our current knowledge, anaerobic reduction of the azo bond by bacteria seems to be better suited for the decolorization of azo dyes in sewage treatment systems. The putative advantages of this method are: ● ● ● The depletion of oxygen is easily accomplished in static cultures and enables anaerobic, facultatively anaerobic, and aerobic bacteria to reduce the azo dyes. The reactions take place at neutral pH values and are expected to be extremely unspecific when low-molecular redox mediators are involved. The reduction rates generally increase in the presence of other carbon sources. The reduction equivalents that are formed during anaerobic oxidation of these carbon sources are finally used for the reduction of the azo bond. The main restriction to the anaerobic treatment of azo compounds is that the amines that are formed are usually not further metabolized under anaerobic conditions (Brown and Hamburger 1987) and there is only one example demonstrating the growth of a (methanogenic) anaerobic consortium on a model azo compound (azodisalicylate) (Razo-Flores et al. 1997). The accumulation of these reduction products from the azo dyes is especially relevant if the amines are presumed carcinogens (e.g. naphthylamine or benzidine derivatives). This problem is of serious concern for human health, because the relevant amines are also formed in the body in the anaerobic compartment of the lower intestine after ingestion of these dyes and may be even formed by skin bacteria (Brown and DeVito 1993; Chung et al. 1992; Platzek et al. 1999). Therefore the relevant dyes have been banned from the market in some countries (e.g. Germany) and the problem may be solved by regulatory efforts (Reife and Freeman 2000). Anaerobic/aerobic treatment of azo dyes Since certain aromatic amines and also sulfonated aminoaromatics are aerobically degraded by bacteria (Brown and Laboureur 1983b; Feigel and Knackmuss 1993; Locher et al. 1989; Nörtemann et al. 1986, 1994; Ohe and Watanabe 1986; Thurnheer et al. 1986, 1988), it has been repeatedly suggested to combine the anaerobic cleavage of the azo dyes with an aerobic treatment 76 Fig. 5A–I Examples of azo compounds that have been studied in anaerobic/aerobic treatment systems. A Mordant Yellow 3; B 4phenylazophenol; C Mordant Yellow 10; D Acid Yellow 17; E Reactive Red 141 (Procion Red H-E7B); F Acid Orange 10; G Acid Red 14; H Acid Red 18; I Reactive Violet 5 (An et al. 1996; Fitzgerald and Bishop 1995; Glässer et al. 1992; Haug et al. 1991; O'Neill et al. 2000a, b; Sosath and Libra 1997; Tan et al. 1999) system for the amines formed. The feasibility of this strategy was first demonstrated for the sulfonated azo dye Mordant Yellow 3 (Fig. 5A) (Glässer et al. 1992; Haug et al. 1991). The anaerobic/aerobic treatment can be carried out either sequentially or simultaneously. Sequential processes may combine the anaerobic and the aerobic step either alternately in the same reaction vessel or in a continuous system in separate vessels (Glässer et al. 1992). The simultaneous treatment systems utilize anaerobic zones within basically aerobic bulk phases, such as observed in biofilms, granular sludge or biomass immobilized in other matrices (Field et al. 1995; Jiang and Bishop 1994; Kudlich et al. 1996; Tan et al. 1999; Zhang et al. 1995). In the sequential and simultaneous treatment systems, auxiliary substrates are required, which supply the bacteria in the anaerobic zones with a source of carbon and energy and a source of reduction equivalents for the cleavage of the azo bond. Although at least certain azo dyes can be mineralized by anaerobic/aerobic treatment systems, also this strategy has serious drawbacks. The fact that many of the amines which are formed during the anaerobic reduction of the azo dyes (which are very often ortho-aminohydroxynaphthalenes) are rather unstable under aerobic conditions and undergo auto-oxidation reactions is a serious problem if a true mineralization of the azo dyes is the aim of the treatment. A recent analysis of these autooxidation reactions suggested that the fate of differently substituted ortho-aminohydroxynaphthalenes varies and that depending on the degree of sulfonation either dimers, disulfonated cinnamic acid derivatives or naphthoquinone imines are formed as major reaction products (Kudlich et al. 1999). It will require further work in order to analyze if a biological degradation of the aminohydroxynaphthalenes can compete with these auto-oxidation reactions and whether the products of the auto-oxidation reactions are accessible for a biological mineralization. During the last few years, different reactor designs have been proposed in order to obtain an effective continuous anaerobic/aerobic treatment of azo dyes: an anaerobic and an aerobic rotating biological contactor (Zaoyan et al. 1992), an anaerobic fixed-film fluidized bed reactor followed by an aerobic suspended-bed activated sludge reactor (Fitzgerald and Bishop 1995; Seshadri et al. 1994), a combination of anaerobic and aerobic rotating-drum reactors (Harmer and Bishop 1992; Sosath and Libra 1997), and an anaerobic up-flow fixed bed column together with an aerobic agitated tank (An et al. 1996; O'Neill et al. 2000a, b; Rajaguru et al. 2000). It is very difficult to compare the efficiencies of these treatment systems because of differences in the dyes and conditions used, the presence of auxiliary carbon sources, and the difficulties in the analysis of the biological or spontaneous reactions of the (auto-oxidizable) amines formed during the anaerobic reactions (for an overview of the azo dyes that have been analyzed in anaerobic/aerobic treatment systems, see Fig. 5). In general, it may be concluded that, in continuous anaerobic/aerobic systems which are fed with substrate mixtures with a high biological and chemical oxygen demand (BOD, COD) and low dye concentrations to the anaerobic stage, a complete decolorization of the dyes and a significant BOD and COD removal can be achieved in the anaerobic stage. In the subsequent aerobic step, the remaining BOD from the auxiliary substrates may be mineralized. There are several examples demonstrating COD removal in the anaerobic/aerobic processes of 70–95% [e.g. for the treatment of Reactive Red 141 (Procion Red H-E7B) (Fig. 5 E) in a simulated textile effluent containing modified starch, O'Neill et al. 2000a, b]. Similar results have also been described for the treatment of wastewater from a dyeing factory on a laboratory scale (Zaoyan et al. 1992). Because the concentrations of the azo dyes are generally much lower than those of the auxiliary substrates, the fate of the aromatic amines formed (especially if they are auto-oxidizable) in the aerobic treatment process is still unclear and some contradicting results have been published. For the treatment of the copper-containing dye Reactive Violet 5 77 (Fig. 5I) in an anaerob/aerobic system with three rotating-disc reactors, no indications for a mineralization of the amines in the aerobic stage were detected by Sosath and Libra (1997). In contrast, the analysis of the fate of nitrogen-containing compounds (presumed amines) in the aerobic step of Reactive Red 141 (Fig. 5E) treatment suggested a decrease in the concentration of nitrogencontaining metabolites (O'Neill et al. 2000a). It is clear that the fate of the reduction products of the azo dyes will vary significantly depending on their tendency to be subject to auto-oxidation processes and/or biodegradation. Preliminary results suggested that the aerobic incubation of certain ortho-aminohydroxynaphthalenes with activated sludge resulted in a reduced number of products formed from the ortho-aminohydroxynaphthalenes compared to the abiotic auto-oxidation processes, and indications for a biological conversion of some products have been found (Kudlich et al. 1999). Thus, encouraging results have been obtained in laboratory experiments, which demonstrated that the anaerobic disintegration of azo dyes results in products that are significantly more available for subsequent aerobic processes. This resulted recently in the decision to build, for the first time, a full-scale anaerobic/aerobic treatment plant for the treatment of wastewater from the textile processing industry. The plant is scheduled to treat more than 1,000 m3 of dye-containing wastewater per day (Krull et al. 2000). References Adamson RH, Dixon RL, Francis FL, Rall DP (1965) Comparative biochemistry of drug metabolism by azo and nitro reductase. Proc Natl Acad Sci USA 54:1386–1391 An H, Qian Y, Gu X, Tang WZ (1996) Biological treatment of dye wastewaters using an anaerobic-oxic system. Chemosphere 33:2533–2542 Anliker R (1979) Ecotoxicology of dyestuffs – a joint effort by industry. Ecotox Environ Safety 3:59–74 Banat IM, Nigam P, Singh D, Marchant R (1996) Microbial decolorization of textile-dye-containing effluents: a review. Biores Technol 58:217–227 Barr DP, Aust SD (1994) Mechanisms white rot fungi use to degrade pollutants. Environ Sci Technol 28:79A-87A Blümel S, Contzen M, Lutz M, Stolz A, Knackmuss H-J (1998) Isolation of a bacterial strain with the ability to utilize the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as sole source of carbon and energy. Appl Environ Microbiol 64:2315–2317 Blümel S, Busse H-J, Stolz A, Kämpfer P (2001a) Xenophilus azovorans gen. nov. sp. nov., a soil bacterium able to degrade azo dyes of the Orange II type. Int J Syst Evol Bacteriol (in press) Blümel S, Mark B, Busse H-J, Kämpfer P, Stolz A (2001b) Pigmentiphaga kullae gen. nov., sp. nov., a new member of the family Alcaligenaceae with the ability to decolorize aerobically azo dyes. Int J Syst Evol Bacteriol (in press) Bragger JL, Lloyd AW, Soozandehfar SH, Bloomfield SF, Marriott C, Martin GP (1997) Investigations into the azo reducing activity of a common colonic microorganism. Int J Pharmaceut 157:61–71 Bromley-Challenor KCA, Knapp JS, Zhang Z, Gray NCC, Hetheridge MJ, Evans MR (2000) Decolorization of an azo dye by unacclimated activated sludge under anaerobic conditions. Water Res 34:4410–4418 Brown D, Hamburger B (1987) The degradation of dyestuffs. Part III. Investigations of their ultimative biodegradability. Chemosphere 16:1539–1553 Brown D, Laboureur P (1983a) The degradation of dyestuffs. Part I. Primary biodegradation under anaerobic conditions. Chemosphere 12:397–404 Brown D, Laboureur P (1983b) The aerobic biodegradability of primary aromatic amines. Chemosphere 12:405–414 Brown J (1981) Reduction of polymeric azo and nitro dyes by intestinal bacteria. Appl Environ Microbiol 41:1283–1286 Brown MA, DeVito SC (1993) Predicting azo dye toxicity. Crit Rev Environ Sci Technol 23:249–324 Bumpus JA (1995) Microbial degradation of azo dyes. In: Singh VP (ed) Microbial degradation of health risk compounds. Elsevier, Amsterdam, pp 157–167 Cao W, Mahadevan B, Crawford DL, Crawford RL (1993) Characterization of an extracellular azo dye-oxidizing peroxidase from Flavobacterium sp. ATCC 39723. Enzyme Microb Technol 15:810–817 Carliell CM, Barclay SJ, Naidoo N, Buckley CA, Mulholland DA, Senior E (1994) Anaerobic decolorisation of reactive dyes in conventional sewage treatment processes. Water SA 20:341–344 Chang J-S, Lin Y-C (2000) Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain. Biotechnol Prog 16:979–985 Chao WL, Lee SL (1994) Decoloration of azo dyes by three white-rot fungi: influence of carbon source. World J Microbiol Biotechnol 10:556–559 Chen K-C, Huang W-T, Wu J-Y, Houng J-Y (1999) Microbial decolorization of azo dyes by Proteus mirabilis. J Ind Microbiol Biotechnol 23:686–690 Chivukula M, Renganathan V (1995) Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl Environ Microbiol 61:4374–4377 Chivukula M, Spadaro JT, Renganathan V (1995) Lignin peroxidase-catalyzed oxidation of sulfonated azo dyes generates novel sulfophenyl hydroperoxides. Biochemistry 34:7765– 7772 Chudgar RJ (1985) Azo dyes. In: Kroschwitz JI (ed) Kirk-Othmer encyclopedia of chemical technology, 4th edn, vol 3. Wiley, New York, pp 821–875 Chung K-T, Fulk GE, Egan M (1978) Reduction of azo dyes by intestinal anaerobes. Appl Environ Microbiol 35:558–562 Chung K-T, Stevens SE, Cerniglia CE (1992) The reduction of azo dyes by the intestinal microflora. Crit Rev Microbiol 18:175–190 Churchley JH (1994) Removal of dyewaste colour from sewage effluent- the use of a full scale ozone plant. Water Sci Tech 30:275–284 Clarke A, Anliker R (1980) Organic dyes and pigments. In: Hutzinger O (ed) The handbook of environmental chemistry, vol 3. Part A. Anthropogenic compounds. Springer, Berlin, Heidelberg, New York, pp 181–215 Contzen M, Moore ERB, Blümel S, Stolz A, Kämpfer P (2000) Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism. Syst Appl Microbiol 23:487–493 Correia VM, Stephenson T, Judd SJ (1994) Characteristics of textile wastewaters – a review. Environ Technol 15:917–929 Coughlin MF, Kinkle BK, Tepper A, Bishop PL (1997) Characterization of aerobic azo dye-degrading bacteria and their activity in biofilms. Water Sci Tech 36:215–220 Coughlin MF, Kinkle BK, Bishop PL (1999) Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX. Ind Microbiol Biotechnol 23:341–346 Cripps C, Bumpus JA, Aust SD (1990) Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl Environ Microbiol 56:1114–1118 Delée W, O′Neill C, Hawkes FR, Pinheiro HM (1998) Anaerobic treatment of textile effluents: a review. J Chem Technol Biotechnol 73:323–335 Dieckhues B (1960) Untersuchungen zur reduktiven Spaltung der Azofarbstoffe durch Bakterien. Zentralbl Bakteriol Parasitenk Infektionskr Abt I Orig 180:244–249 78 Dohányos M, Madera V, Sedlácek M (1978) Removal of organic dyes by activated sludge. Prog Water Technol 10:559–575 Dubin P, Wright KL (1975) Reduction of azo food dyes in cultures of Proteus vulgaris. Xenobiotica 5:563–571 Dykes GA, Timm RG, von Holy A (1994) Azoreductase activity in bacteria associated with the greening of instant chocolate puddings. Appl Environ Microbiol 60:3027–3029 Feigel BJ, Knackmuss H-J (1993) Syntrophic interactions during degradation of 4-aminobenzenesulfonic acid by a two species bacterial culture. Arch Microbiol 159: 124–130 Field JA, Stams AJM, Kato M, Schraa G (1995) Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia. Antonie van Leeuwenhoek 67:47–77 Fitzgerald SW, Bishop PL (1995) Two stage anaerobic/aerobic treatment of sulfonated azo dyes. J Environ Sci Health A 30:1251–1276 Ganesh R, Boardman GD, Michelsen D (1994) Fate of azo dyes in sludges. Water Res 28:1367–1376 Ghosh DK, Mandal A, Chaudhuri J (1992) Purification and partial characterization of two azoreductases from Shigella dysenteriae type 1. FEMS Microbiol Lett 98:229–234 Ghosh DK, Ghosh S, Sadhukhan P, Mandal A, Chauduri J (1993) Purification of two azoreductases from Escherichia coli K12. Indian J Exp Biol 31:951–954 Gill M, Strauch RJ (1984) Constituents of Agaricus xanthodermus Genevier: The first naturally endogenous azo compound and toxic phenolic metabolites. Z Naturforsch 39c:1027–1029 Glässer A, Liebelt U, Hempel DC (1992) Design of a two-stage process for total degradation of azo dyes. DECHEMA Biotechnol Conf 5B:1085–1088 Glenn JK, Akileswaran L, Gold MH (1986) Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 251:688–696 Goszczynski S, Paszczynski A, Pasti-Grigsby MB, Crawford RL, Crawford DL (1994) New pathway for degradation of sulfonated azo dyes by microbial peroxidases of Phanerochaete chrysosporium and Streptomyces chromofuscus. J Bacteriol 176:1339–1347 Hardin IR, Cao H, Wilson SS, Akin DE (2000) Decolorization of textile waste water by selective fungi. Textile Chemist and Colorist and American Dyestuff Reporter 32:38–42 Harmer C, Bishop P (1992) Transformation of azo dye AO-7 by wastewater biofilms. Water Sci Technol 26:627–636 Haug W, Schmidt A, Nörtemann B, Hempel DC, Stolz A, Knackmuss H-J (1991) Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium. Appl Environ Microbiol 57:3144–3149 Hausser A (1995) Abbau von 4,4′-Dicarboxyazobenzol durch einen neu isolierten Bakterienstamm. Studienarbeit Universität Stuttgart Hayase N, Kouno K, Ushio K (2000) Isolation and characterization of Aeromonas sp. B-5 capable of decolorizing various dyes. J Biosci Bioeng 90:570–573 Heinfling A, Bergbauer M, Szewzyk U (1997) Biodegradation of azo and phtalocyanine dyes by Trametes versicolor and Bjerkandera adusta. Appl Microbiol Biotechnol 48:261–266 Heinfling A, Martinez MJ, Martinez AT, Bergbauer M, Szewzyk U (1998) Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese independent reaction. Appl Environ Microbiol 64:2788–2793 Hernandez PH, Gilette R, Mazel P (1967a) Studies on the mechanism of action of mammalian hepatic azoreductase. I. Azoreductase activity of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochem Pharmacol 16: 1859–1875 Hernandez PH, Gilette R, Mazel P (1967b) Studies on the mechanism of action of mammalian hepatic azoreductase. II. The effect of phenobarbital and 3-methylcholanthrene on carbon monoxide sensitive and insensitive azoreductase activity. Biochem Pharmacol 16:877–1888 Hitz HR, Huber W, Reed RH (1978) The adsorption of dyes on activated sludge. JCDC 94:71–76 Horitsu H, Takada M, Idaka E, Tomoyeda M, Ogawa T (1977) Degradation of p-aminoazobenzene by Bacillus subtilis. European J Appl Microbiol 4:217–224 Idaka E, Ogawa T, Horitsu H, Tomoyeda M (1978) Degradation of azo compounds by Aeromonas hydrophila var. 24B. JSDC 94:91–94 Jeckel M (1997) Wastewater treatment in the textile industry (1997) In: Kornmüller A (ed) Treatment of wastewaters from textile processing. Schriftenreihe Biologische Abwasserreinigung 9, TU Berlin, pp 3–12 Jiang H, Bishop PL (1994) Aerobic biodegradation of azo dyes in biofilms. Water Sci Tech 29:525–530 Keck A, Klein J, Kudlich M, Stolz A, Knackmuss H-J, Mattes R (1997) Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation of Sphingomonas sp. strain BN6. Appl Environ Microbiol 63:3684–3690 Kim SJ, Ishikawa K, Hirai M, Shoda M (1995) Characteristics of a newly isolated fungus, Geotrichum candidum Dec 1, which decolorizes various dyes. J Ferment Bioeng 79:601–607 Kim SJ, Shoda M (1999) Purification and characterization of a novel peroxidase from Geotrichum candidum Dec 1 involved in decolorization of dyes. Appl Environ Microbiol 65:1029– 1035 Knapp JS, Newby PS, Reece LP (1995) Decolorization of dyes by wood-rotting basidiomycete fungi. Enzyme Microb Technol 17:664–668 Krull R, Hempel DC, Metzen P, Alfter P (2000) Konzept zur technischen Umsetzung einer zweistufigen anoxischen und aeroben Textilabwasserbehandlung. Chem-Ing-Tech 72:1113–1114 Kudlich M, Bishop P, Knackmuss H-J, Stolz A (1996) Synchronous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow 3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture. Appl Microbiol Biotechnol 46: 597–603 Kudlich M, Keck A, Klein J, Stolz A (1997) Localization of the enzyme system involved in the anaerobic degradation of azo dyes by Sphingomonas sp. BN6 and effect of artificial redox mediators on the rate of azo reduction. Appl Environ Microbiol 63:3691–3694 Kudlich M, Hetheridge MJ, Knackmuss H-J, Stolz A (1999) Autoxidation reactions of different aromatic ortho-aminohydroxynaphthalenes which are formed during the anaerobic reduction of sulfonated azo dyes. Environ Sci Technol 33:896–901 Kulla HG (1981) Aerobic bacterial degradation of azo dyes. In: Leisinger T, Cook AM, Nüesch J, Hütter R (eds) Microbial degradation of xenobiotics and recalcitrant compounds. Academic, London pp 387–399 Kulla HG, Klausener F, Meyer U, Lüdeke B, Leisinger T (1983) Interference of aromatic sulfo groups in the microbial degradation of the azo dyes Orange I and Orange II. Arch Microbiol 135:1–7 Kulla HG, Krieg R, Zimmermann T, Leisinger T (1984) Experimental evolution of azo dye-degrading bacteria. In: Klug MJ, Reddy CA (eds) Current perspectives in microbial ecology. American Society of Microbiology, Washington, DC, pp 663–667 Libra J, Yoo ES, Borchert M, Wiesmann U (1997) Mechanism of biological decolorization: their engineering application. In: Kornmüller A (ed.) Treatment of wastewaters from textile processing. Schriftenreihe Biologische Abwasserreinigung 9, TU Berlin, pp 245–266 Locher HH, Thurnheer T, Leisinger T, Cook AM (1989) 3-Nitrobenzenesulfonate, 3-aminobenzenesulfonate, and 4-aminobenzenesulfonate as sole carbon sources for bacteria. Appl Environ Microbiol 55:492–494 Maguire RJ, Tkacz RJ (1991) Occurence of dyes in the Yamaska River, Québec. Water Poll Res J Canada 26:145–161 Mechsner K, Wuhrmann K (1982) Cell permeability as a rate limiting factor in the microbial reduction of sulfonated azo dyes. Eur J Appl Microbiol Biotechnol 15:123–126 79 Mecke R, Schmähl D (1957) Die Spaltbarkeit der Azo-Brücke durch Hefe. Arzneim-Forsch 7:335–340 Namasivayam C, Yamuna RT (1992) Removal of Congo Red from aqueous solutions by biogas waste slurry. J Chem Tech Biotechnol 53:153–157 Nörtemann B, Baumgarten J, Rast HG, Knackmuss H-J (1986) Bacterial communities degrading amino- and hydroxynaphthalenesulfonates. Appl Environ Microbiol 52:1195–1202 Nörtemann B, Kuhm AE, Knackmuss H-J, Stolz A (1994) Conversion of substituted naphthalenesulfonates by Pseudomonas sp. BN6. Arch Microbiol 161:320–327 Ogawa T, Yatome C, Idaka E, Kamiya H (1986) Biodegradation of azo acid dyes by continuous cultivation of Pseudomonas cepacia 13NA. JSDC 102:12–14 Ohe T, Watanabe Y (1986) Degradation of 2-naphthylamine-1-sulfonic acid by Pseudomonas strain TA-1. Agric Biol Chem 50: 1419–1426 Ollikka P, Alhonmäki K, Leppänen V-M, Glumoff T, Raijola T, Suominen I (1993) Decolorization of azo, triphenyl methane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Appl Environ Microbiol 59: 4010–4016 O’Neill C, Lopez A, Esteves S, Hawkes FR, Hawkes DL, Wilcox S (2000a) Azo-dye degradation in an anaerobic-aerobic treatment system operating on simulated textile effluent. Appl Microbiol Biotechnol 53:249–254 O’Neill C, Hawkes FR, Hawkes DL, Esteves S, Wilcox SJ (2000b) Anaerobic-aerobic biotreatment of simulated textile effluent containing varied ratios of starch and azo dye. Water Res 34:2355–2361 Overney G (1979) Ueber den aeroben Abbau von Dicarboxyazobenzol durch ein Flavobacterium sp. PhD Thesis Diss ETH 6421, ETH Zürich, Switzerland Pagga U, Brown D (1986) The degradation of dyestuffs. Part II. Behaviour of dyestuffs in aerobic biodegradation tests. Chemosphere 15:479–491 Pagga U, Taeger K (1994) Development of a method for adsorption of dyestuffs on activated sludge. Water Res 28:1051–1057 Panswed J, Wongehaisuwan S (1986) Mechanism of dye waste water color removal by magnesium carbonate-hydrate basic. Water Sci Technol 18:139–144 Pasti-Grigsby MB, Paszczynski A, Goszczynski S, Crawford DL, Crawford RL (1992) Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium. Appl Environ Microbiol 58:3605–3613 Pasti-Grigsby MB, Burke NS, Goszczynski S, Crawford DL (1996) Transformation of azo dye isomers by Streptomyces chromofuscus A11. Appl Environ Microbiol 62:1814–1817 Paszczynski A, Crawford RL (1991) Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol. Biochem Biophys Res Comm 178:1056–1063 Paszczynski A, Pasti MB, Goszczynski S, Crawford DL, Crawford RL (1991) New approach to improve degradation of recalcitrant azo dyes by Streptomyces spp. and Phanerochaete chrysosporium. Enzyme Microb Technol 13:378–384 Paszczynski A, Pasti-Grigsby MB, Goszczynski S, Crawford RL, Crawford DL (1992) Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. Appl Environ Microbiol 58: 3598–3604 Perlinger J, Angst W, Schwarzenbach RP (1996) Kinetics of reduction of hexachloroethane by juglone in solutions containing hydrogen sulfide. Environ Sci Technol 30:3408–3417 Platzek T, Lang C, Grohmann G, Gi U-S, Baltes W (1999) Formation of carcinogenic aromatic amine from an azo dye by human skin bacteria in vitro. Human Exp Toxicol 18:552–559 Rafii F, Franklin W, Cerniglia CE (1990) Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. Appl Environ Microbiol 56:2146–2151 Rafii F, Moore JD, Ruseler-van Embden JGH, Cerniglia CE (1995) Bacterial reduction of azo dyes used in foods, drugs and cosmetics. Microecol Ther 25:147–156 Ràfols C, Barceló D (1997) Determination of mono- and disulphonated azo dyes by liquid chromatography-atmospheric pressure ionization mass spectrometry. J Chromatogr A 777:177–192 Rajaguru P, Kalaiselvi K, Palanivel M, Subburam V (2000) Biodegradation of azo dyes in a sequential anaerobic-aerobic system. Appl Microbiol Biotechnol 54:268–273 Razo-Flores E, Luijten M, Donlon BA, Lettinga G, Field JA (1997) Complete biodegradation of the azo dye azodisalicylate under anaerobic conditions. Environ Sci Technol 31:2098– 2103 Reife A, Freeman HS (2000) Pollution prevention in the production of dyes and pigments. Text Chem Color & Am Dyes Rep 32:56–60 Reisch MS (1996) Asian textile dye makers are a growing power in changing market. Chem Eng News Jan 15:10–12 Riu J, Schönsee I, Barceló D (1998) Determination of sulfonated azo dyes in groundwater and industrial effluent by automated solid-phase extraction followed by capillary electrophoresis/mass spectrometry. J Mass Spectrom 33:653–663 Rodríguez E, Pickard MA, Vazquez-Duhalt R (1999) Industrial dye decolorization by laccases from lignolytic fungi. Curr Microbiol 38:27–32 Roxon JJ, Ryan AJ, Wright SE (1967) Enzymatic reduction of tartrazine by Proteus vulgaris from rats. Fd Cosmet Toxicol 5:645–656 Russ R, Rau J, Stolz A (2000) The function of cytoplasmatic flavin reductases in the bacterial reduction of azo dyes. Appl Environ Microbiol 66:1429–1434 Scheline RR, Nygaard RT, Longberg B (1970) Enzymatic reduction of the azo dye, Acid Yellow, by extracts of Streptococcus faecalis, isolated from rat intestine. Food Cosmet Toxicol 8:55–58 Schliephake K, Mainwaring DE, Lonergan GT, Jones IK, Baker WL (2000) Transformation and degradation of the disazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus. Enzyme Microb Technol 27:100–107 Schönberger H (1997) Modern concepts for reducing the wastewater load in the textile processing. In: Kornmüller A (ed.) Treatment of wastewaters from textile processing. Schriftenreihe Biologische Abwasserreinigung 9, TU Berlin, pp 13–24 Schwarzenbach RP, Stierli R, Lanz K, Zeyer J (1990) Quinone and iron porphyrine mediated reduction of nitroaromatic compounds in homogenous aqueous solution. Environ Sci Technol 24:1566–1574 Seshadri S, Bishop PL, Agha AM (1994) Anaerobic/aerobic treatment of selected azo dyes in waste water. Waste Management 14:127–137 Shaul GM, Holdsworth TJ, Dempsey CR, Dostal KA (1991) Fate of water soluble azo dyes in the activated sludge process. Chemosphere 22:107–119 Shin K-S, Kim C-J (1998) Decolorisation of artificial dyes by peroxidase from the white-rot fungus, Pleurotus ostreatus. Biotechnol Lett 20:569–572 Sosath F, Libra JA (1997) Biologische Behandlung von synthetischen Abwässern mit Azofarbstoffen. Acta hydrochim hydrobiol 25:259–264 Spadaro JT, Gold MH, Renganathan V(1992) Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium. Appl Environ Microbiol 58: 2397–2401 Spadaro JT, Renganathan V (1994) Peroxidase-catalyzed oxidation of azo dyes: Mechanism of Disperse Yellow 3 degradation. Arch Biochem Biophys 312:301–307 Sugiura W, Miyashita T, Yokoyama T, Arai M (1999) Isolation of azo-dye degrading microorganisms and their application to white discharge printing of fabric. J Biosci Bioeng 88:577– 581 Swamy J, Ramsay JA (1999a) The evaluation of white rot fungi in the decoloration of textile dyes. Enzyme Microb Technol 24:130–137 Swamy J, Ramsay JA (1999b) Effects of glucose and NH +4 concentrations on sequential dye decoloration by Trametes versicolor. Enzyme Microb Technol 25:278–284 80 Tan, N, Prenafeta-Boldú FX, Opsteeg JL, Lettinga G, Field JA (1999) Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures. Appl Microbiol Biotechnol 51:865–871 Thurnheer T, Köhler T, Cook AM, Leisinger T (1986) Orthanilic acid and analogues as carbon sources for bacteria: growth physiology and enzymic desulphonation. J Gen Microbiol 132:1215–1220 Thurnheer T, Cook AM, Leisinger T (1988) Co-culture of defined bacteria to degrade seven sulfonated aromatic compounds: efficiency, rates and phenotypic variations. Appl Microbiol Biotechnol 29:605–609 Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26 Tincher WC, Robertson JR (1982) Analysis of dyes in textile dyeing wastewater. Text Chem Color 14:269–275 Walker R (1970) The metabolism of azo compounds: a review of the literature. Food Cosmet Toxicol 8: 659–676 Weber EJ, Stickney VC (1993) Hydrolysis kinetics of Reactive Blue 19-vinyl sulfone. Water Res 27:63–67 Wong PK, Yuen PY (1996) Decolorization and biodegradation of methyl red by Klebsiella pneumoniae RS-13. Water Res 30:1736–1744 Wuhrmann K, Mechsner K, Kappeler T (1980) Investigation on rate-determining factors in the microbial reduction of azo dyes. Eur J Appl Microbiol 9:325–338 Yatome C, Matsufuru H, Taguchi T, Ogawa T (1993) Degradation of 4′-dimethylaminoazobenzene-2-carboxylic acid by Pseudomonas stutzeri. Appl Microbiol Biotechnol 39:778–781 Yatome C, Ogawa T, Koda D, Idaka E (1981) Biodegradability of azo and triphenylmethane dyes by Pseudomonas pseudomallei 13NA. JSDC 97:166–169 Yeh RYL, Thomas A (1995) Color difference measurement and color removal from dye wastewaters using different adsorbents. J Chem Tech Biotechnol 63:55–59 Yoo ES, Libra J, Wiesmann U (1999) Untersuchungen zum anaeroben Entfärbungsmechanismus von Azofarbstoffen. DECHEMA Jahrestagung: 399–400 Yoshida H, Fukuda S, Okamoto A, Kataoka T (1991) Recovery of direct dye and acid dye by adsorption on chitosan fiber-equilibria. Water Sci Technol 23:1667–1676 Young L, Yu J (1997) Ligninase-catalysed decolorization of synthetic dyes. Water Res 31:1187–1193 Zaoyan Y, Ke S, Guangliang S, Fan Y, Jinshan D, Huanian M (1992) Anaerobic-aerobic treatment of a dye wastewater by combination of RBC with activated sludge. Water Sci Tech 26:2093–2096 Zbaida S (1995) The mechanism of microsomal azoreduction: predictions based on electronic aspects of structure-activity relationships. Drug Metab Rev 27:497–516 Zhang F, Knapp JS, Tapley KN (1999) Development of bioreactor systems for decolorization of Orange II using white rot fungus. Enzyme Microb Technol 24:48–53 Zhang TC, Fu YC, Bishop PL, Kupferle M, FitzGerald S, Jiang HH, Harmer C (1995) Transport and biodegradation of toxic organics in biofilms. J Hazard Mat 41:267–285 Zheng Z, Levin RE, Pinkham JL, Shetty K (1999) Decolorization of polymeric dyes by a novel Penicillium isolate. Proc Biochem 34:31–37 Zimmermann T, Kulla HG, Leisinger T (1982) Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur J Biochem 129:197– 203 Zimmermann T, Gasser F, Kulla HG, Leisinger T (1984) Comparison of two azoreductases acquired during adaptation to growth on azo dyes. Arch Microbiol 138:37–43 Zissi U, Lyberatos G, Pavlou S (1997) Biodegradation of p-aminoazobenzene by Bacillus subtilis under aerobic conditions. J Ind Microbiol Biotechnol 19:49–55 Zollinger H (1991) Color chemistry, 2nd edn., VCH, Weinheim