Decolorization and Degradation of Textile Dyes with Biosulfidogenic Hydrogenases C. C. Z. Mutambanengwe, C. A. Togo, and C. G. Whiteley* Department of Biochemistry, Microbiology, and Biotechnology, Rhodes University, Grahamstown 6140, South Africa Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480- 620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds. 1. Introduction Enzyme technology in industrial wastewater bioremediation is superior to the redundant chemical and physical procedures because there is a limited production of hazardous byproducts, it is relatively cheap if whole cells are used, there is a low energy requirement, and it is environmentally friendly. With particular reference to the textile industry, dyes are classified as an- thraquinone, phthalocyanine, triphenylmethane, heterocyclic, and azo, with the last mentioned representing one of the largest class used and characterized by the functional azo group (-NdN-). These brightly colored dyes are water-soluble and are extremely resistant to microbial and physicochemical degradation, including conventional processes of wastewater treatment. The microbiological decolorization of industrial effluent wastewater containing these dyes is ongoing with an increasing number of studies being reported (1-10). The loss in color, for a typical azo dye, is due to a reduction of the azo group first to the bis-amine then to two separate amines. These dyes and their degradative aromatic amine products are toxic and mutagenic to living systems and consequently are undesir- able if released into the environment. Enzymes from both anaerobic and aerobic systems have been reported to be effective in the decolorization of the dyes (11-23), with the majority of results forthcoming from white rot fungi Phanaerochaete and Trametes and yeasts (19-23). These organisms secrete laccases and manganese/lignin peroxidases that are capable, by a biocatalytic process, of oxidative free radical cleavage of the azo bond. Furthermore, it has been suggested that nonspecific cytoplasmic reductases and hydrogenases from certain bacteria may also degrade azo dyes (24, 25). It is hypothesized that an anaerobic degradation of the dyes into their constituent aromatic amines followed by an aerobic degradation into CO 2 ,H 2 O, and NH 3 takes place. Due to the variability in industrial effluent composition as well as the structural diversity of the dye itself, the biodegradation of azo dyes, specifically sulfonated azo dyes, found in the textile sectors are more recalcitrant to aerobic biodegradation (13, 26). As far as we are aware the complete degradation of the azo dye aromatic ring system has been successful only by a sequence of anaerobic-aerobic processes, and no reports are forthcoming of a degradation solely by an anaerobic process. Since hydrogenase enzymes have been found within an anaerobic biosulfidogenic reactor in our laboratories (27, 28) it was decided to investigate their role in the bioremediation of industrial textile dye effluents. This paper reports on the decolorization and degradation of various azo and non-azo textile dyes from both industrial and commercial sources by their incubation in a biosulfidogenic reactor along with hydrogenase enzymes that are present in situ. 2. Materials and Methods 2.1. Materials. TRIS HCl, methyl viologen, Orange II, Amido Black 10, Reactive Black 5, Reactive Red 120, and Reactive Blue 2 were obtained from Sigma-Aldrich (South Africa). Snakeskin dialysis tubing was purchased from Amersham Biosciences (South Africa). Analytical-grade reagents were used * To whom correspondence should be addressed. Tel: +27-46-6038085. Fax: +27-46-6223984. Email: c.whiteley@ru.ac.za.