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สารฮิ วมิ กช่วยเพิ่ มความสามารถการลดสี ... 198 The Journal of the Royal Institute of Thailand Vol. 37 No. 1 Jan.-Mar. 2012 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 characterization 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 0 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 l -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 (OD600) of approximately 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 concentration = 100 µM) 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 centrifugation (10,000 x g , 10 min), and the concentration of sulfonated azo dyes in supernatant was determined spectrophotometrically at λ max of each azo dye.