Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis

Abstract

The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damköhler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35-38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis.