Most industrial fermentation processes are operated in fed-batch mode to overcome catabolite repression, undesired by-product formation and oxygen limitation. To maintain comparable process conditions during screening of optimal production strains, the implementation of a fed-batch mode at small scale is crucial. In this study, three different protease producing Bacillus species, Bacillus aeolius, B. licheniformis and B. pumilus, were cultivated using the previously described membrane-based fed-batch shake flasks. Under carbon-limited conditions, catabolite repression was avoided, so that proteases were produced in all strains. Protease yields of B. aeolius and B. licheniformis increased 1.5-fold relative to batch cultivations. To validate process scalability between shake flasks and stirred tank reactors, membrane-based fed-batch shake flask cultivations were transferred to laboratory-scale stirred tank reactors with equal feeding rates. Despite inevitable differences between the scales such as pH control, feed supply and feed start, comparable results were achieved. Oxygen transfer rates of B. licheniformis and B. pumilus measured with the respiration activity monitoring system (RAMOS) in shake flasks and in stirred tank reactor with an off-gas analyzer were almost identical in both cultivation systems. The protease activities referring to the total consumed glucose were also mostly comparable. A slight decrease from shake flask to stirred tank reactor could be observed, which is presumably due to differences in pH control. This study successfully demonstrates the transferability of membrane-based fed-batch shake flask cultivations to laboratory-scale stirred tank reactors. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.