Oxidative Degradation of Azo Dyes by Manganese Peroxidase under Optimized Conditions I. Mielgo, C. Lo ´ pez, M. T. Moreira, G. Feijoo, and J. M. Lema* Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. The goal of this study is the development of a degradation system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) for the degradation of azo dyes. The experimental work also includes the optimization of the process, with the objective of determining the influence of specific physicochemical factors, such as organic acids, H 2 O 2 addition, Mn 2+ concentration, pH, temperature, enzyme activity and dye concentration. A nearly total decolorization was possible at very low reaction times (10 min) and at high dye concentration (up to 1500 mg L -1 ). A specific oxidation capacity as high as 10 mg dye degraded per unit of MnP consumed was attained for a decolorization higher than 90%. Among all, the main factor affecting process efficiency was the strategy of H 2 O 2 addition. The continuous addition at a controlled flow permitted the progressive participation of H 2 O 2 in the catalytic cycle through a suitable regeneration of the oxidized form of the enzyme, which enhanced both the extent and the rate of decolorization. It was also found that, in this particular case, the presence of a chelating organic acid (e.g., malonic) was not required for an effective operation. Probably, Mn 3+ was chelated by the dye itself. The simplicity and high efficiency of the process open an interesting possibility of using of MnP for solving other environmental problems. Introduction In recent years the massive increase in the synthesis of chemicals has led to the production of a wide variety of compounds, some of which are xenobiotic or persistent in natural ecosystems. Synthetic dyes belong to this type of compounds; most of them have an aromatic structure, which makes them highly stable and thus difficult to degrade (1). Nowadays, systems based on physicochem- ical treatments (flocculation, ozonation or membrane filtration) have currently been used for the treatment of effluents containing dyes (2). However, these treatments produce an accumulation of sludges, which will eventu- ally have to be treated. Conventional biological waste- water treatment systems, such as activated sludges, are ineffective in dye removal because most of these com- pounds pass through these plants without any structural modification. Legislation relating to colored effluent discharges is becoming stricter, and new technologies are needed to solve this environmental problem. White-rot fungi may constitute an upcoming alterna- tive for the degradation of dyes and other recalcitrant compounds because they produce oxidases and per- oxidases, which are highly oxidative and substrate- nonspecific. The most outstanding enzymes are lignin peroxidase (LiP) and manganese peroxidase (MnP), first described in Phanerochaete chrysosporium (3, 4). The oxidation of dyes by fungal cultures has been made possible (5, 6); however, the major limitation associated with their application relates to the time necessary to carry out the degradation (7). From a practical point of view, the use of peroxidases in vitro for that purpose may represent a more feasible system, provided that a number of conditions are ful- filled: (a) a simple and applicable system; (b) minimal requirements of compounds; (c) low cost and stable enzyme; and (d) short treatment periods. This system could be implemented as a pre- or as a post-treatment for conditioning those concentrated fractions of effluents with strong color. There are several studies of in vitro incubations of azo dyes with crude or purified LiP (8-11) or MnP (12). The assays reported were performed on a very small scale, and only a limited decolorization yield was achieved. This low efficiency may be due to either some compounds lacking in the amounts required or to the nonoptimized physicochemical conditions, both of which are required for the enzymatic action. The main objective of this paper is the evaluation of the use of MnP from Bjerkandera sp. BOS55 for the degradation of azo dyes. This enzyme presents high versatility since it has both MnP and LiP substrate interaction sites (7, 13), and in this way, it arises as an interesting alternative to develop an in vitro degradation system. The action of MnP depends on the combined action of several compounds, referred to as substrates, cofactors and mediators, which initiate, participate in, and allow the completion of the catalytic cycle. Therefore, special attention was paid to study the influence of the * To whom correspondence should be addressed. Tel: 34-981- 563100, Ext. 14231. Fax: 34-981-595012. E-mail: jmlema@usc.es. 325 Biotechnol. Prog. 2003, 19, 325-331 10.1021/bp020136w CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/10/2003