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«“√ “√ √“™∫— ≥±‘ µ¬ ∂“π ªï ∑’Ë Û ©∫— ∫∑’Ë Ò ¡.§.-¡’ .§. ÚıÙ¯ 59 Somsak Damronglerd, et al. degradation by aerobic bacteria due to the strong electron-withdrawing property of the azo group thought to protect against attack by oxygenases (28) so that there generally pass biodegradative processes in conven- tional sewage treatment systems untreated (40, 48). The azo dyes- containing effluents from these industries have caused serious environment pollution, because the presence of dyes in water is highly visible and affects their transparency and aesthetics even if the concentra- tion of the dyes is low (23). Microbial decolorization of sulfonated azo dyes readily occurs under anaerobic conditions by a wide variety of bacteria utilizing several intracellular reductases to reductive cleavage of the azo dyes to produce corresponding colorless aromatic amines (9, 13, 19, 22, 44, 45). These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the microorganisms involved and the dyes converted. In the textile processing industry, a wide range of structurally diverse dyes is used within short time periods in one and the same factory and, therefore, the effluents from the industry are markedly variable in composition (17). Moreover, in the case of sulfonated azo dyes, sulfonic acid substitutions seem to be an effective inhibitor of permeation of the dyes through the cell membrane (56) and therefore, intracellular reductases do not function (46). Thus, an extra- cellular nonspecific biological process may be vital for treatment of the textile effluents. From the currently known biological systems, the required unspecifity may be obtained by using the suitable redox mediator system. The unspecific anaerobic reduction of azo compounds very often low-molecular-weight redox mediators (e.g., flavins or quinones) are involved (14, 26, 29, 42, 43, 50). These mediators are reduced by bacterial enzymes to corresponding their reduced forms (e.g., reduced flavins or hydroquinones) which enable the transfer of redox equivalents to extracellular azo dyes in a purely chemical reaction (14, 42, 50). These alternative redox mediators either occur naturally in groundwater and sediments or are possible additives for stimulating in situ biodecolori- zation processes.Also, organic matter in the natural environment may contain humic substances, which are known to accelerate reductive processes by redox mediation (7, 11, 31, 47). In the present study, humic substance is evaluated as a potential redox mediator for the reduction of sulfonated azo dyes. Humic substance is the stable organic matter accumulating in soils and sediments (49). Although humic substance is generally consider to inert for microbial catabolism, it has recently been reported to play an active role in anaerobic oxidation of a wide variety of ecologically relevant organic substrate (10, 15, 31). These studies have demonstrated that the reduc- tion of humic substances may be important mechanisms for organic substrate oxidation inmany anaerobic environments. Quinone moieties of humus have been implicated as redox active groups (47) accepting the electrons. Anthraquinone-2, 6-disulfonate (AQDS) have been used as a defined model for such moieties (10, 11, 31). Most AQDS- respiring microorganisms are capable of transferring electrons to AQDS, reducing it to anthraquinone-2,6- disulfonate (AH 2 QDS). The role of humic analog AQDS as an electron shuttle has been demonstrated previously and thought to provide a strategy for Fe (III) reducers to access insoluble Fe (III) compounds (31). The anaerobic microbial oxidation of phenol and p -cresol in granular sludge was recently found to be coupled to the reduction of AQDS (11). The addition of humic acids or AQDS was also shown to stimulate the mineralization of the priority pollutants vinyl chloride and dichlo- roethene by a humus-respiring consortiumunder anaerobic conditions (7). The rates of azo dye decoloriza- tion are also enhanced in the presence of different quinoid redox mediators, especially anthraquinone-2-sulfonate. (29, 42, 59). Because the utilization of humic substance as a redoxmediator should allowvery unspecific reduction processes with various azo dyes, in the present study it was therefore to examined the mechanism which, humic substance stimulate anaerobic reduction of sulfonated azo dyes by Paenibacillus sp. strain A5. The location of the enzyme systemwhich is responsible for the reduction of sulfonated azo dyes by whole cells of strainA5 in the presence of humic