Enzyme and Microbial Technology 31 (2002) 848–854 Improved conditions for the aerobic reductive decolourisation of azo dyes by Candida zeylanoides Patricia A. Ramalho a,∗ , H. Scholze b , M. Helena Cardoso a , M. Teresa Ramalho b , A.M. Oliveira-Campos b a Department of Biology, University of Minho, Braga, Portugal b Department of Chemistry, University of Minho, Braga, Portugal Received 16 January 2002; received in revised form 19 July 2002; accepted 23 July 2002 Abstract A number of anaerobic and aerobic bacterial species are known to decolourise azo dyes through the reduction of the azo bonds, forming the corresponding amines. In this work, we describe improved decolourisation conditions for model azo dyes by the ascomycete yeast Candida zeylanoides. The dyes were derived from the diazonium salts of metanilic and sulfanilic acids and N,N-dimethylaniline or 2-naphthol as coupling components. Total decolourisation times observed in culture media supplemented with 0.2 mM dye ranged from 40 to 60 h. The initial decolourisation rates were 14–52 mol (g dry cell) -1 h -1 , depending on dye structure. In the course of decolourisation either metanilic acid or sulfanilic acid were detected in the supernatant fluid, showing that decolourization by this yeast strain is due to azo bond reduction. None of those aminobenzenesulfonates supported microorganism growth as carbon and energy source but both could be used, to a limited extent, as nitrogen sources. The azo reductase activity is not significantly affected by pre-adaptation of the microorganism to the dyes. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Azo dyes; Methyl orange; Orange II; Yeasts; Decolourisation 1. Introduction Among the synthetic dyes, which are widely used for textile dyeing and other industrial applications, those con- taining an azo chromophore constitute the largest class [1]. Since dyes are designed to be resistant to microbial and physico-chemical attack, most of them are not easily destroyed by conventional processes of wastewater treat- ment, including biological treatment by activated sludge [2,3]. The percentage of unchanged dye after such treat- ments is estimated to range between 50 and 90% [4,5], a fact which raises environmental problems, not only for obvious aesthetic reasons but also because the dyes themselves, or their biotransformation products, can have deleterious effects on living organisms. Both the structural diversity of dyes and the wide variability in composition of dye-containing effluents are probably decisive factors accounting for the observed recalcitrance of colouring substances [6]. ∗ Corresponding author. Tel.: +351-253-604-386; fax: +351-253-678-983. E-mail address: tramalho@quimica.uminho.pt (P.A. Ramalho). As reviewed by Chung et al. [7], earlier studies on the decolourisation of azo dyes mainly involved anaerobic bac- terial species isolated from the intestinal microflora, which cleave the molecules by reduction of the azo bond(s). A closely related research field deals with the investigations on the toxic, mutagenic, or carcinogenic properties of several aromatic amines generated by biological reduction [8,9]. The azo reductase activity detected in bacteria displaying azo bond reduction capabilities has also deserved considerable attention. Azo reductases were isolated from several bacte- rial sources and some of their molecular properties and sub- strate specificities were determined [10,11]. Some authors have also succeeded in isolating genomic DNA fragments from azo-reducing bacterial species and expressing them in non-decolourising species [12–14]. The available evidence indicates that azo reductase activity is associated with dif- ferent types of genes [12]. In other words, azo reductase activity can be associated with more than one reductase, de- pending on the particular microorganism and, eventually, on the cultivation conditions. The most generally accepted mechanism of azo reduc- tion involves the participation of redox mediators, acting as electron shuttles between the extracellular dye and the 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00189-8