สำนักราชบัณฑิตยสภา

The Journal of the Royal Institute of Thailand Vol. 30 No. 1 Jan.-Mar. 2005 60 Humic Substance Enhanced Anaerobic Reduction of Sulfonated Azo Dyes by Paenibacillus sp. Strain A5 substance was also determined. MATERIALS AND METHODS Bacteria strain and culture condition. Paenibacillus sp. strain A5, a facultative anaerobic and endospore- forming bacteria, was originally isolated from textile effluent treatment plant. The isolation and characteri- zation of this strain have been described before (51). Cell suspensions of strain A5 are able to anaerobically reduce various sulfonated azo dyes to corresponding aromatic amines (52). Strain A5 was routinely grown at 30˚C on minimal medium containing glucose as a sole carbon and energy source. The basic composition of minimal medium (MMG) was (in g l -1 ): glucose 0.9, (NH 4 ) 2 SO 4 0.28, NH 4 Cl 0.23, KH 2 PO 4 0.067, MgSO 4 • 7H 2 O 0.04, CaCl 2 • 2H 2 O 0.022, FeCl 3 • 6H 2 O 0.005, NaCl 0.15, NaHCO 3 1.0 and 1 ml 1 -1 of a trace element solution containing (in g l -1 ) ZnSO 4 • 7H 2 O 0.01, MnCl 2 • 4H 2 O 0.1, CuSO 4 • 5H 2 O 0.392, CoCl 2 • 6H 2 O 0.248, NaB 4 O 7 • 10H 2 O 0.177 and NiCl 2 • 6H 2 O 0.02. Anaerobic reduction of sulfonated azo dyes with whole cells. Cells of strain A5 were grown aerobically in MMG to an optical density at 600 nm (OD 600 )of appro- ximately 1. Then cells were harvested by centrifugation, washed twice, and resuspended in 50 mM Na-K phosphate buffer (pH 7.5). The cell suspension (protein concentration of approximately 0.1 g l -1 ) was transferred into screw cap-glass tubes (10 ml). The reaction mixture contained in a final volume of 10 ml, 5 mM of glucose, 50 mM of Na-K phosphate buffer (pH 7.5), and different concentration of redox mediators. Oxygen was removed from reaction mixture by evacuation and flushing with oxygen-free nitrogen gas for 5 min. The reaction was started by the addition of sulfonated azo dyes (final concentra- tion = 100 M) from anaerobic stock solution into reaction mixture. To prevent possible contamination with oxygen during sampling, tubes were opened only once, and only as many glass tubes were incubated as measurements were planned. The cells were removed by centrifuga- tion (10,000 g, 10 min), and the concentration of sulfonated azo dyes in supernatant was determined spectrophotometrically at λ max of each azo dye. Standard assay for determination of azo reducatase activities with cell extracts and soluble cell membrane in the presence of different redox mediators. The azo reductase activity was determined anaerobically in 1.5 ml-rubber-stoppered cuvettes which were flushed before the assay with oxygen-free nitrogen gas. Azo reductase activity was routinely measured by a modification of the spectrophotometric assay described previously by Kudlich et al. (29) and Russ et al. (46). For the standard assay, the anaerobically prepared reaction mixtures contained in 800 l of 50 Mof Tris-HCl buffer (pH 7.5), 25 M of the respective sufonated azo dyes, and 50 M of redox mediators (FAD or AQDS). The cell extracts or solubilized cell membrane from Paenibacillus sp. strainA5 were added (200 l, about 0.05 to 0.1 mg of protein), and the reaction mixtures were flushed again with nitrogen gas. Finally, the reaction was started by the addition of 300 M of NADH, and the initial rate was measured at 30˚C with a model UVWINLAB, Perkin Elmer molecular spectrophoto- meter for 30 min (by using 1-min measuring intervals). Reaction rates were calculated by usingmolar extinc- tion coefficient of these azo dyes, which are summarized in Table 1. Preparation of cell membranes. StrainA5 was grown aerobically under the conditions described above until they reached the late exponential growth phase. Cells were harvested by centrifugation at 10,000 g for 10 min, washed twice, and resuspended in Na-K phosphate buffer (50 mM, pH 7.5) to an OD 600 of about 5. Cell-free extracts and cell membranes were prepared by disruption of a suspension of whole cells (protein content, about 1 g l -1 ) by ultrasonication using an ultrasonic processor (Sonicator ® W-385, Heat systems-Ultrasonics, Inc.). After removing the cell debris by centrifuga- tion at 10,000 g for 15 min at 4˚C, the supernatant was ultracentrifuged at 100,000 g for 30 min at 4 ˚C (29, 43). The supernatant of ultracentrifu- gation was used in certain experiment as the “cytoplasmic fraction”. In the same time, the transparent pellet