During the last decades single-use bioreactors have become widely accepted in the biopharmaceutical industry. Single-use technologies bring many advantages over conventional solutions, such as a reduced investment and operational cost as well as an optimized time-to-market. So far, this type of bioreactor is mainly used for cell culture applications. Results for microbial fermentations are rarely found, commonly due to limited oxygen transfer rates. The aim of this study was to establish a high cell density fermentation process for a recombinant Escherichia coli considering a rocking motion type and an adapted stirred 50-L single-use bioreactor. By using the design space approach an OTRmax/OURmax model was established. The feeding strategies were optimized and verified based on a model for both single-use systems to achieve high cell densities. In both single-use bioreactors a recombinant protein was successfully expressed.