Improvement of the production of subtilisin Carlsberg alkaline protease by Bacillus licheniformis by on-line process monitoring and control in a stirred tank reactor

The cultivation of Bacillus licheniformis and the production of subtilisin Carlsberg serine protease were investigated in complex medium with starch and glucose, respectively, and Na-caseinate as substrates to maximize the protease concentration. The turbidity and culture fluorescence were monitored in situ, the optical density on-line and the (dry) sediment and (dry) cell mass concentrations as well as the cell count and the DNA content were monitored off-line. These values are closely interrelated and were quantified by particular relationships. By means of the six-channel flow injection analyser (FIA) system, the following medium components were monitored on-line: glucose, maltose, starch, ammonium, urea, phosphate and protease activity. The same components as well as protein, intracellular phosphate and-α-amylase activity were evaluated off-line. The off-gas composition was analysed on-line as well. Various control strategies were tested in order to maximize the protease concentration: On one hand, starch in various concentrations was used as substrate. These runs were performed at non-controlled starch decomposition, at controlled and non-controlled pH-values, respectively, and non-controlled Po2-values. On the other hand, glucose was used as substrate in fed-batch mode. These runs were performed with closed loop controlled pH- and Po2-values and open-loop and closed-loop controlled glucose concentrations, respectively. The latter strategy yielded a higher protease concentration than the former. With complex medium and closed-loop controlled process, extremely high protease activities (24 000 EPE ml −1) were obtained.