Bioprocess Parameters and Oxygen Transfer Characteristics in -Lactamase Production by Bacillus Species

After screening potential (beta)-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) (beta)-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH4)2HPO4, NH4Cl, yeast extract, casamino acids and peptone, pH, and temperature on (beta)-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm^-3 in the medium containing 10.0 kg m^-3 glucose, 1.18 kg m^-3 (NH4)2HPO4, 8.0 kg m^-3 yeast extract, and the salt solution at 32° C and pH0 = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm^3 bioreactor systems with a VR = 1.65 dm^3 working volume at QO/VR = 0.5 vvm and N = 500 min^-1. At the beginning of the process the Damkohler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >>1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (KLa) varied between 0.01 and 0.03 s^-1, enhancement factor (KLa/KLaO) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m^-3 s^-1 throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as YX/S = 0.34 and YX/O = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as YS/O = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as mO = 0.13 kg kg^-1 h^-1 and mS = 3.02 kg kg^-1 h^-1.