Effects of pH on metabolic flux distributions in P-lactamase producing Bacillus licheniformis were investigated using metabolic flux analysis, at pH(C) = 6.5, 7.0 and 7.5 controlled-pH conditions. The bioprocess was divided into three periods: Period I (0 < t < 6 h) is the growth phase where beta-lactamase synthesis starts; Period II (6 < t < 8 h) is the transition phase; Period III (8 < t < 12 h) is the stationary phase for the cell-growth where beta-lactamase synthesis increases. The central pathways were active for the growth and beta-lactamase synthesis throughout the bioprocess at all the conditions. In Period I, the fiuxes through the by-products were the highest at pHC = 7.0 while the lowest at pH(C) = 7.5; however, there was no significant difference between the beta-lactamase synthesis fluxes. On the other hand, in Period III the by-product and lactamase fluxes were the highest at pH(C) = 7.5 and 7.0, respectively. Based on the intracellular-reaction-network fluxes to increase beta-lactamase production further the following pH operation strategy is proposed: initial pH of the fermentation medium should be set to pH = 7.5, and pH of the medium can be let to decrease until pH = 7.0; thereafter, controlled at pH(C) = 7.0 until the end of the bioprocess. (c) 2007 Elsevier Ltd. All rights reserved.