Enzyme and Microbial Technology 46 (2010) 360–365 Contents lists available at ScienceDirect Enzyme and Microbial Technology j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e m t Polymerization study of the aromatic amines generated by the biodegradation of azo dyes using the laccase enzyme Elisangela Franciscon a,∗ , Francine Piubeli a , Fabiana Fantinatti-Garboggini c , Cristiano Ragagnin de Menezes a , Isis Serrano Silva a , Artur Cavaco-Paulo b , Matthew James Grossman d , Lucia Regina Durrant a a Campinas State University, Department of Food Science, 13083-970 Campinas, São Paulo, Brazil b University of Minho, Department of Textile Engineering, 4800-058 Guimarães, Portugal c Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), Campinas State University, São Paulo, Brazil d BioSage – 807 Eagles Chase Drive, Lawrenceville, NJ 08648, United States a r t i c l e i n f o Article history: Received 18 September 2009 Received in revised form 18 December 2009 Accepted 21 December 2009 Keywords: Azo dyes Biodegradation Bacteria Aromatic amines Polymerization Laccase a b s t r a c t Four different azo dyes were decolorized (color reduction >90%) by bacteria isolated from a textile wastewater effluent. Dye decolorizing was carried out under microaerobic conditions until completion, after which the aromatic amine concentration was determined.A laccase from Myceliophthora ther- mophila was used to catalyze coupling reactions of the aromatic amines produced from decolorizing the dyes. The reaction was carried out with stirring (100 rpm) in a weak acidic buffer solution (pH 5.0) at 45 ◦ C for 3 days. The presence of aromatic amines in the samples after bacterial decolorizing confirmed the azo bond was reduced in the process. In addition, the UV–vis spectrum was shifted significantly after the sequential bacterial-laccase treatment also indicating a chemical transformation of the dyes. After laccase treatment the solutions formed colored soluble and precipitated products. The particles sizes making up the precipitates formed after laccase treatment varied between 105 and 483 nm as deter- mined by Photon Correlation Spectroscopy (PCS). The laccase treatment also reduced the COD of the dye solutions by ∼20%. We show that successive bacterial-laccase treatment is effective in decolorized azo dyes by reduction of the azo bonds,and promoting coupling reactions between the aromatic amines formed. Promoting coupling reactions between the aromatic amines using enzymes may prove useful for the physical removal and reuse of these amines. © 2010 Published by Elsevier Inc. 1. Introduction Azo dyes constitute the largest and most versatile class of syn- thetic dyes used in the textile, pharmaceutical, food and cosmetics industries. These dyes are characterized by the presence of one or more azo bonds (–N N–). These are prepared by azo coupling between a diazonium compound and an aniline,phenol or other aromatic compound. During the dyeing process in the textile indus- try, approximately 10–15% of the dyes used are released into the wastewater. Aromatic amines, with known carcinogenic potential, such as aniline and sulphanilic acid appear in liquid effluents as a conse- quence biological reduction of the azo bond in azo dyes [1].The presence of these dyes and their by-products in aqueous ecosys- tems leads to aesthetic and health problems due to the coloring ∗ Corresponding author. Tel.: +55 19 3521 2173; fax: +55 19 35212153. E-mail address: elisfran@fea.unicamp.br (E. Franciscon). of waters, the obstruction of light penetration and inhibition of oxygen transfer [2,3]. Dye wastewaters are usually treated by flocculation, coagu- lation, adsorption, membrane filtration,precipitation, irradiation, ozonization and Fenton’s oxidation [4].These methods are often expensive and can generate large amounts of sludge, which increases process costs [5].The use of biological methods in the treatment of textile wastewaters may be a cost effective alterna- tive to the physico-chemical and photochemical methods currently used. Aerobic biological methods are largely ineffective in the treat- ment of textile wastewaters, resulting in little or no color removal in the case of most of the dyes, especially azo dyes. Anaerobic and microaerobic treatments are effective in removing the color, but the products from azo dye degradation are frequently carcino- genic aromatic amines and these amines can inhibit further aerobic degradation[6,7]. Conventional processes for the removal of aromatic amines from industrial wastewaters include extraction, adsorption onto acti- vated carbon, bacterial and chemical oxidation, electrochemical 0141-0229/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2009.12.014