Polysialic acid (polySia), consisting of α-(2,8)-linked N-acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave-induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h−1. Investigation of stress-related product release from the cell surface showed no significant differences between the disposable bag reactor with wave-induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L−1 and a polySia concentration of 245 mg L−1 were reached. The total protein concentration in the supernatant was 132 mg L−1. After efficient and time-saving downstream processing characterization of the final product showed a protein content of below 0.04 mgprotein/gpolySia and a maximal chain length of ∼90 degree of polymerization. This is the peer reviewed version of the following article: de Vries, I.; Busse, C.; Kopatz, J.; Neumann, H.; Beutel, S.; Scheper, T.: Polysialic acid production using Escherichia coli K1 in a disposable bag reactor. In: Engineering in Life Sciences (2017), which has been published in final form at https://doi.org/10.1002/elsc.201600220. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.