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Azo dyes are a widespread class of poorly biodegradable industrial pollutants. In anaerobic environments, azo bonds are reductively cleaved yielding carcinogenic aromatic amines, many of which are assumed to resist further metabolism by anaerobes. Here we report for the first time that an azo dye compound is completely biodegradable in the absence of oxygen. A pharmaceutical azo dye, azodisalicylate, constructed from two 5-aminosalicylic acid (5ASA) molecules was mineralized in an adapted methanogenic consortium to CH4 and NH3 with transient accumulation of 5ASA as a degradation intermediate in both batch assays and continuous bioreactors. The anaerobic metabolism of 5ASA was shown to provide the electrons required for the initial reductive cleavage of the azo group. Our results suggest that it is possible to design azo dyes that are fully mineralized in the environment; thereby, avoiding ac cumulation of notorious toxic intermediates.

Biological treatment of wastewaters discharged by the textile industry could potentially be problematic due to the high toxicity and recalcitrance of the commonly-used azo dye compounds. In the present report, the fate of two azo dyes under methanogenic conditions was studied. Mordant Orange 1 (MO1) and Azodisalicylate (ADS) were completely reduced and decolorised in continuous UASB reactors in the presence of cosubstrates. In the MO1 reactor, both 5-aminosalicylic acid (5-ASA) and 1,4-phenylenediamine were identified as products of azo cleavage. After long adaptation periods, 5-ASA was detected at trace levels, indicating further mineralization. ADS, a pharmaceutical azo dye constructed from two 5-ASA units, was completely mineralized even in the absence of cosubstrate, indicating that the metabolism of 5-ASA could provide the reducing equivalents needed for the azo reduction. Batch experiments confirmed the ADS mineralization. These results demonstrate that some azo dyes could serve as a carbon, energy, and nitrogen source for anaerobic bacteria.

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H 2 lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H 2 lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.Methanogenic decomposition of complex organic matter is a widespread process, which accounts for a large fraction of the global methane emission (15). Examples of natural methanogenic habitats are freshwater environments such as wetlands, sediments, and rice paddy fields, as well as intestinal tracts of higher animals and insects (4,6,8,21,28). Methanogenic processes can be applied to treat industrial wastewaters in high-rate anoxic bioreactors (16,19,33). The microorganisms involved in methanogenic decomposition are usually immobilized in granular aggregates or biofilms, which is essential for a high conversion rate (23) and prevents biomass from being washed out of the reactor.The amount of energy available in methanogenic conversions is small, and therefore the microorganisms involved are forced to cooperate syntrophically (24, 28). In particular, oxidation of intermediary reduced organic compounds, such as ethanol, butyrate, and propionate, is energetically unfavorable. Nevertheless, the methanogens involved keep the concentrations of the oxidation products, acetate and H 2 (or formate), low enough to create a situation in which all partners involved gain energy. To dispose of reducing equivalents, acetogens reduce protons or bicarbonate. Both H 2 and formate have been proposed as electron carriers in syntrophic degradation. Several studies have provided evidence for H 2 transfer by demonstrating syntrophic growth with methanogens that oxidize only H 2 . Schmidt and Ahring (25) studied interspecies electron transfer in granules from a mesophilic anaerobic sludge bed reactor, and concluded that formate transfer was not important during propionate and butyrate oxidation in this system.On the other hand, Thiele and Zeikus (29) provided evidence that interspecies formate transfer was the dominant mechanism in a whey-processing digester,...

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