ORIGINAL PAPER B. Donlon · E. Razo-Flores · M. Luijten · H. Swarts G. Lettinga · J. Field Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor Received: 11 July 1996 / Received revision: 18 September 1996 / Accepted: 18 September 1996 Abstract In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methano- genic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards me- thanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was in- vestigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no co- substrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was re- ductively cleaved to less toxic aromatic amines (1,4- phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as co- substrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sam- pled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane. Introduction Azo dyes are one of the oldest industrially synthesized organic compounds. The azo dye production in the United States in 1985 amounted to over 10 8 kg, which was used, by textile, printing, drug and pharmaceutical industries (United States International Trade Commis- sion 1986). Approximately 10 000 dyes are currently manufactured (Zollinger 1987) and it is estimated that at least 15% of these are released into the environment (Vaidya and Datye 1982). They occur in industrial ef- fluent, groundwater, contaminated soils and sediments. These compounds are of concern because some of the dyes, dye precursors or their biotransformation pro- ducts, such as the aromatic amines, have been shown to be carcinogenic (Kriek 1979; Longstaff 1983). Azo dyes are designed to be recalcitrant under typical product service conditions and it is this property, allied with their toxicity to microorganisms, that makes biological treatment difficult (Idaka et al. 1985; Ogawa et al. 1978). Biological treatment systems may have promising applications for the removal of azo dye compounds since it is widely reported that the azo dyes are gratuitously reduced by anaerobic sludges, anaerobic sediments and anaerobic bacterial enrichment cultures (Brown and Hamburger 1987; Brown and Laboureur 1983a; Chung et al. 1992; Weber and Wolfe 1987). On the other hand, azo dye compounds are resistant to oxygenolytic attack. Pagga and Brown (1986) and Shaul et al. (1991) tested the degradation of more than 100 dyes in aerobic acti- vated-sludge systems and found that only a few of them were actually biodegraded. Appl Microbiol Biotechnol (1997) 47: 83 – 90  Springer-Verlag 1997 B. Donlon 1 · E. Razo-Flores (&) · M. Luijten · G. Lettinga J. Field Department of Environmental Technology, Wageningen Agricultural University, Bomenweg 2, 6700 EV Wageningen, The Netherlands. Fax: 31 317 482108 e-mail: Elias.Razo@algemeen.mt.wau.nl E. Razo-Flores Instituto Mexicano del Petro ´ leo, Subdireccio ´ n de Proteccio ´ n Ambiental, Eje Central La ´zaro Ca ´rdenas 152, 07730 Me ´xico, D.F., Me ´xico H. Swarts Department of Organic Chemistry, Wageningen Agricultural University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands Present address: 1 EPA Headquarters, Ardcavan, Wexford, Ireland