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The Journal of the Royal Institute of Thailand Vol. 30 No. 1 Jan.-Mar. 2005 62 Humic Substance Enhanced Anaerobic Reduction of Sulfonated Azo Dyes by Paenibacillus sp. Strain A5 Cells were harvested by centrifuga- tion for 10 min at 10,000 g after the cultures reached an OD 600 ≈ 2.0. Pellets were resuspended in 6 ml chloroform: methanol (2 : 1, v/v) and the suspension was gently mixed overnight in complete darkness at room temperature. The suspension was filtered though Whatman no. 1 filter paper and filtrate was evaporated under reduced pressure. The residue was resuspended in ethyl acetate, and solution was spotted on Silica Gel 60 F254 aluminum-packed TLC plates (E. Merck, Germany). Menaquinone (MK-1) and ubiquinone (Q1) were used as standards. Samples were eluted with a mixture of n-hexane and diethyl ether (85:15, v/v). Quinones were detected on TLC plates by brief irradiation with short-wave ultraviolet light (33). Analytical techniques The concentration of sulfonated azo dyes was determined spectropho- tometrically (UV WINLAB, Perkin Elmer). Sulfonated azo dyes and the corresponding reduction products were analyzed by high performance liquid chromatography (HPLC; Shimadzu model LC-3A chroma- tograph (Shimadzu Corp., Kyoto, Japan) equipped with Shimadzu model SPD-2A detector). A reverse- phase column was Pegasil ODS, (4.6 mm x 150 mm [inside diameter] column, Senshu Scientific Co., Ltd., Tokyo, Japan). A mobile phase composed of 50% methanol, 0.3% H 3 PO 4 , and 49.7% water was used with the flow rate of 0.5 ml min -1 . The eluates were monitored by UV absorption at 275 nm. Concentra- tion of AH 2 QDS were determined- spectrophotometrically by monitoring the absorbance at 450 nm by using a molar extinction coefficient of 2.7 mM -1 cm -1 obtained from a calibration curve of AQDS chemically reduced by dithionite as previously described (10). Chemicals. Remazol Brilliant Orange 3R, Remazol Black B, and Remazol Brilliant Violet 5R were kindly supplied by Dystar, Thailand, Ltd. NADH, FAD, ubiquinone (Q1), and menaquinone (MK) were purchased form Sigma Chemical Co., St. Louis, Mo. AQDS, riboflavin, and humic acid were purchased from Aldrich Chemical (Milwaukee, Wis.). All other chemicals used for minimum medium and buffer solutions were obtained from E. Merck AG. (Darmstadt, Germany) and Aldrich Chemical (Milwaukee, Wis.). RESULTS AQDS stimulation of different sulfonated azo dyes reduction by whole cells of strain A5. To determine whether AQDS stimulated bacterial azo dyes reduc- tion, cell suspensions of azo dyes- reducing bacterium Paenibacillus sp. strain A5 were added to phosphate buffer containing glucose as the electron donor and various sulfonated azo dyes (for structural formulas see Fig. 1) and were incubated under anaerobic condition. Cell suspen- sions of strainA5 only slowly reduced azo dyes in the absence of additional redox mediator, but when the AQDS was also added to the buffer, azo dyes reduction was greatly stimulated (Fig. 2). The observed variations in the relative increase of the azo dye reduction rates for different dyes were presumably due to differences in the redox potentials of the individual azo compounds. The lesser but significant stimulation was observed in this study with the commercially available Aldrich humic acids that was previously found to stimulate the dissimilatory reduction of ferric iron (31). Abiotic reduction of sulfonated azo dyes did not occur in controls without cells or added electron donor (Fig. 2). Quinone component(s) in cell membrane of strain A5. Newman andKolter have recent- ly shown that there is a common biochemical basis for AQDS and humic acid respiration involving the biosynthesis of menaquinone, a lipophilic naphthoquinone with a prenyl side chain of variable length that participates in the transfer of electrons to low-potential electron acceptors and normally found within the lipid bilayer of cell membrane (34). Every Bacillus sp. so far exam- ined contains menaquinone as the sole quinone component and general absence of ubiquinone (33). Like all the other bacilli, lipid extraction of culture grown aerobically showed that only menaquinone was found in Paenibacillus sp. strain A5. (as assayed by thin layer chromatography). Thin layer chromatography also revealed that menaquinone was not present in the supernatants of strain A5.