192 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2005, The Society for Biotechnology, Japan Vol. 100, No. 2, 192–196. 2005 DOI: 10.1263/jbb.100.192 Photodecolorization of Azo Dyes by Extracellular Metabolites under Fluorescent Light and Influence of Operational Parameters Jinglan Hong, 1 * Hiroko Emori, 1 and Masahiro Otaki 1 Department of Human Environmental Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan 1 Received 31 January 2005/Accepted 26 April 2005 The contribution of extracellular metabolites (EM) to the decolorant activity of newly isolated photosynthetic bacteria was observed. The decolorization process was considered to occur by two paths: photochemical decolorization by EM and photobiological decolorization by photosynthetic bacteria. In addition, the decolorization of several azo dyes by EM under black light and fluores- cent light irradiation was investigated. It was found that EM were capable of decolorizing the azo dyes directly under visible light irradiation, and the overall dye decolorization followed first- order decay kinetics. Moreover, the decolorization reaction is a nonenzymatic reaction and the unknown metabolite with the decolorizing ability had an apparent molecular weight lower than 3 kDa as determined by ultrafiltration. In addition, its decolorization activity was stable even after heating sterilization at 121°C for 10 min. Furthermore, the decolorization rate increased with increasing optical intensity, temperature and EM concentration, and decreased with increas- ing initial dye concentration. Decolorization of dye was best at pH 8. [Key words: extracellular metabolites, decolorization, azo dye, visible light irradiation] Azo dyes are the largest chemical class of dyes regularly used for textile dyeing, color printing, color photography and other industrial applications, due to their highly economic feasibility in synthesis, firmness, and variety of colors com- pared to natural dyes (1). During the last two decades, color removal from textile industry wastewater has attracted con- siderable attention due to the color visibility in treated water and the toxicity of certain dyes. Various physical and chem- ical methods for the treatment of dye-containing water are available, which involve flocculation (2), sorption (3), and electrochemical and oxidative degradation (4, 5) techniques. However, an efficient, safe and cost-effective system for effluent decolorization has not been established. A newly isolated photosynthetic bacterium was used to investigate the removal of dyes in batch systems (6) and continuous systems (7–9). It was found to efficiently decolorize various azo dyes. 16S rRNA and phylogenetic analyses were per- formed to characterize the photosynthetic dye-degrading bacterium and the strain was identified as Rhodobacter sphaeroides (Kuroki, S., Ph.D. thesis, Kumamoto Univer- sity, 2002). In the above-mentioned treatment of dyes, the decoloriza- tion phenomenon of dyes by extracellular metabolites (EM) isolated from the newly isolated photosynthetic bacterium was observed. The objective of our study was to character- ize the azo-dye-decolorizing activity of these EM. MATERIALS AND METHODS Extracellular metabolites Photosynthetic bacterium was isolated according to Kuroki et al. (6). The composition of the pho- tosynthetic bacterial medium used to culture the isolated bacterium in this study was 0.5 g/l K 2 HPO 4 , 0.5 g/l KH 2 PO 4 , 0.2 g/l MgSO 4 - 7H 2 O, 5.3 × 10 –2 g/l CaCl 2 -2H 2 O, 1.0 × 10 –3 g/l thiamine hydrochlo- ride, 1.0 × 10 –3 g/l nicotinic acid, 1.0 × 10 –5 g/l biotin, 1.2 × 10 –3 g/l MnSO 4 -5H 2 O, 2.4 × 10 –3 g/l ferric citrate, 1.0 × 10 –3 g/l CoCl 2 - 6H 2 O, 1.0 × 10 –3 g/l Na 2 MoO 4 -2H 2 O, 2.7 g/l L(-)-malic acid, 3.8 g/l L-glutamic acid monosodium salt and 0.8 g/l (NH 4 ) 2 HPO 4 (10). The isolated photosynthetic bacterium was grown in sterilized glass bottles at 37°C under 3 klx illumination for 15–30 d, and cul- ture media containing the photosynthetic bacterium were centri- fuged at 3000 × g for 10 min. Supernatants were filtered through 0.45-μm membrane filters (Dismic-25cs; Roshi, Tokyo), and the resulting cell-free filtrates containing the metabolites at different concentrations were collected and used as EM. Decolorization experiments The azo dyes acid blue 92 (AB92), acid black 1 (AB1) and reactive black 5 (RB5) were used in this study. The molecular structures of these dyes are shown in Fig. 1. Stock solutions of the appropriate dyes were diluted in deionized water and the pH was adjusted using phosphate buffer solution. Decolorization experiments were conducted in a total volume of 25 ml (in 50-ml glass tubes) in a low-temperature incu- bator (BITEC-300; Shimadzu, Kyoto) under black light (BL; FL20s-BLB; Toshiba, Tokyo) and fluorescent light (FL; FL20SD-B; Hitachi, Tokyo) irradiation. The solutions were continuously stir- red using a magnetic stirrer. Control tubes contained photosyn- thetic bacterial medium without EM. To understand the effects of EM on the decolorant activity of the isolated photosynthetic bac- terium, a series of AB92 decolorant experiments was undertaken. * Corresponding author. e-mail: jlhong@cc.ocha.ac.jp phone/fax: +81-(0)3-5978-5747