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The Journal of the Royal Institute of Thailand Vol. 30 No. 1 Jan.-Mar. 2005 66 Humic Substance Enhanced Anaerobic Reduction of Sulfonated Azo Dyes by Paenibacillus sp. Strain A5 site in several bacterial genera (18, 29, 42, 43, 50, 57). NDH play their most important role as a primary dehydrogenase, linked with the central metabolism, in the respiratory chain of all organisms having an aerobic or anaerobic electron- transport system. Several types of NDH occur in bacteria, the most common of which are referred to as NDH-1 and NDH-2 (16). NDH-1 is an energy-transducing enzyme meanwhile NDH-2 appears to have no role in energy transduction (16). Some gram-positive bacteria such as Bacillus subtilis have non-energy generating NDH 2 but not NDH 1 (3). In addition, it has recently suggested that oxygen-insensitive nitroreductase (NfsA and NfsB) are capable of effectively reducing not onlynitrocompoundsbut alsoquinones, which may not be natural substrates (60). The nitroreductase, and other enzymes in different families, which had sequence homologies to a certain group of NfsA and NfsB such as flavin reductase (FRP) (61), may be also able to function under anaerobic conditions in the appropriate condi- tions as quinone-dependent azo reductases in many bacterial genera (43, 60). Indeed, the further study is necessary to identification the real enzyme system which is responsible for the ability of Paenibacillus sp. strain A5 to reduce AQDS and thus to reduce sulfonate azo dyes under anaerobic condition in the presence of AQDS or other quinoid mediators. In biological systems, quinones were also shown to accelerate azo dye reduction by anaerobically incubated aerobic biomass as well as granular sludge (29, 42, 43). Theoretically, feasible quinoid redox mediators should have redox between those of the two eventual half reactions, the reduction of azo dyes and the oxidation of a primary electron donor. Although standard redox potential (E o ’) for the reduction of the sulfonated azo dyes to their constituent aromatic amines are not available, an indication can be derived from paragraphic data. Redox poten- tials of the azo compounds are approximately vary between -180 mV and -430 mV (19, 42). For bacterial azo dye reduction, i.e., coupled to the oxidation of organic primary electron donors by anaero- bically incubated bacteria, the E o ’ value of NAD(P)H, the cellular redox cofactors with the lowest electron potential (-320 mV), can be taken into account (54). It was recently suggested that quinoid redox media- tors with standard redox potentials (E o ’) between approximately -320 mV and -50 mV could in general function as effective redox mediators in the bacterial reduction of sulfonated azo dyes (42). Thus quinones with a rather negative redox potential such as AQDS are suitable as redox mediators for the anaerobic treatment of azo dyes. Kudlich et al. (29) localized a quinoid redox mediator-dependent azo reductase activity in themembrane of a gram-negative bacterium, Sphin- gomonas sp. strain BN6. Therefore, in the present study, the AH 2 QDS is suggested to shuttle reduction equivalents in the cells to extracellular sulfonated azo dye and reduce the azo compound in a purely chemical reaction (Fig. 5). The anaerobic reduction of AQDS occurred only in the presence of glucose, which seems to be the source of reduction equivalents to reduce the AQDS outside the membrane of strain A5. The proposed mechanism demon- strated in Fig. 5 could be useful in the treatment of sulfonated azo dyes containing wastewaters by strain A5 or other bacteria which able to reduce AQDS. Under anaerobic conditions, the chemical reactions of the reduced AQDS (AH 2 QDS) with the sulfonated azo dyes allows for extremely unspecific reduction processes which are mainly govern- ed by the redox potentials of the AH 2 QDS and azo compounds therefore, a wide range of azo dyes can be reduced. The main restriction to this mechanism is that the amines that are formed are usually not further metabolized under anaerobic condi- tions. Since aromatic amines and also sulfonated aromatics are aerobically degraded by bacteria (8, 20, 37, 35, 36), it has been repeatedly suggested to combined the anaerobic reduc- tion of the azo dyes with an aerobic treatment system for the amines formed (21, 24, 38, 39, 52, 53). The abilityof quinone compounds (i.e., AQDS) to convoy electrons between the bacterial membrane and the dye in solution at the same distance from the cell suggests that Paenibacillus sp. strain A5-AQDS couple can be used to reduce dye in a separate compartment without