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The Journal of the Royal Institute of Thailand Vol. 30 No. 1 Jan.-Mar. 2005 64 Humic Substance Enhanced Anaerobic Reduction of Sulfonated Azo Dyes by Paenibacillus sp. Strain A5 other hand, in earlier studies with facultatively anaerobic bacteria, it was repeatedly suggested that the reduced flavins generated by cytosolic flavin-dependent reductases (flavin reductases) were responsible for unspecific reduction of azo dyes (22, 45, 46). Because quinone reductases and flavin reductase are hypothesized involving the unspecific reduction of azo dyes by bacteria, therefore, in this study we attempted to determine the activity of these enzymes present inmembrane and cytoplasmic fractions of strain A5. For strain A5, it has shown that the high level of NADH: quinone oxidoreductase activity was found in membrane fraction meanwhile the high level of NAD(P) H: flavin oxidoreductase activity was found in cytosolic fraction of the cells (Table 3). Mechanism which AQDS enhance sulfonated azo dyes reduction. The proposed mechanism by which AQDS enhance reduction of azo compounds encloses two independent reactions: first, the quinones are enzymatically reduced to the corresponding hydroquinones, and second, the hydroquinones cleave the azo dyes in a purely chemical reaction (31, 42, 50). Therefore, both reactions were analyzed separately (Fig. 4). For the first reaction, the experimental results shown that the anaerobic Paenibacillus sp. strainA5 suspensions could drive a large fraction ofAQDS to the reduced state (anthrahydroquinone-2,6-disulfonate, AH 2 QDS) as evidenced by the orange color formation and increased absorbance at 450 nm (10)) of the culture medium within 10-h incuba- tion period (Fig. 4). At the end of anaerobic incubation, the buffer turned orange owing to the accumulation of AH 2 QDS. The fact that the introduc- tion of oxygen at the end of the anaerobic incubation resulted in immediate loss of orange color and decreased absorbance confirmed that AQDS was enzymatically reduced during the anaerobic incubation with strain A5 (data not shown). To evaluate the second reaction in proposed mechanism, sulfonated azo dyes were added into cell-free culture filtrates containing ofAH 2 QDS, it was found that the orange color of AH 2 QDS disappeared immediately. Furthermore, it is evident that the amounts of three sulfonated azo dyes reduced rapidly and Remazol Brilliant Orange 3R and Remazol Brilliant Violet 5R were completely removed within a few minutes after addition into filtrates of 10-h anaerobic incubated cell suspensions containing 0.25 mM AQDS. In the other hand,AQDS not incubated with Paenibacillus sp. strain A5 did not reduced all sulfonated azo dyes and filtrates of cell suspensions that did not contain AQDS did not reduced all azo dyes. The addition of fixed concentra- tion of all sulfonated azo dyes (0.1 mM) into the cultured-filtrates containing known concentration of AH 2 QDS ( ≈ 0.25 mM) demonstrated that the chemical reduction of orange and violet dye required two moles of AH 2 QDS to produce the correspond- ing aromatic amines and by four moles of AH 2 QDS to produce the corresponding aromatic amines of black dye.The stoichiometry suggested a complete reduction of sulfonated azo dyes to the corresponding aromatic amines. Characterization of products formed from chemical reduction of various sulfonated azo dyes. The reduction products of sulfonated azo dyes present in the reaction mixtures were analyzed by HPLC as described in Materials and Methods. HPLC chromatogram of reduction products of individual azo dyes byAH 2 QDS was compared with theHPLCchromatogramof completely reduction products of each azo dye obtained by reduction the dye with sodium dithionite, a strong chemical reducing agent (2). Form comparison results, the complete reduction of each azo dye to its corresponding amines byAH 2 QDS was proposed in this study (data not shown). DISCUSSION Several different mechanisms have been proposed for reduction or degradation of azo dyes and similar compounds. A description of a nonspecific azo reductase system involved in azo dye reduction has been provided for selected bacterial species, and it has been shown that the relevant gene is relatively conserved in various anaerobic and facultative bacteria (29, 41). In this research it was also hypothesized that coenzyme reducing equivalents (e.g., NADH) involved in normal electron transport through oxidation of organic