A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain, Bacillus sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80°C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO 2 substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.In the last few years, environmental legislation, e.g., about the appearance of color in discharges, combined with the increasing cost of water for the industrial sector, has made the treatment and reuse of dyeing effluents increasingly attractive to the industry. The common method for the treatment of wastewater in the textile finishing industry is physicochemical flocculation in combination with biological treatment (17). The conventional treatment of colored effluents produces a lot of sludge, but does not remove all dyes, thus preventing recycling of the treated wastewater. In activated sludge treatments, dyeing effluents, e.g., reactive azo dyes and naphthaline-sulfonic acids as well as aromatic amino derivatives, represent an extensive nonbiodegradable class of compounds (18) and can even inhibit activated sludge organisms. Thus, the development of more effective biological systems for the treatment of these types of wastewater has resulted in considerable interest by the industry.Wood-rotting fungi (e.g., Phanerochaete chrysosporium, Trametes sp., and Aspergillus sp.) have been found to effectively degrade a variety of azo dyes under aerobic conditions (16). Dye-degrading fungi have been used frequently in bioreactors for the decolorization and degradation of azo dyes (37). While fungal treatment of effluents is usually very time-consuming (3), immobilized enzymes could have potential for dye decolorization and recycling of effluents (1) without the need for the addition of growth substrates. The enzymes involved in the degradation of azo dyes are mainly peroxidases (12) and laccases (1). However, a significant cost reduction for these enzymes would be required in order to make this process economically more attractive. Aerobic bacteria have been described to oxidatively decolorize m...

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