Diffusion-driven fed-batch fermentation in perforated ring flasks

Simultaneous membrane-based feeding and monitoring of the oxygen transfer rate shall be introduced to the newly established perforated ring flask, which consists of a cylindrical glass flask with an additional perforated inner glass ring, for rapid bioprocess development.

A 3D-printed adapter was constructed to enable monitoring of the oxygen transfer rate in the perforated ring flasks. Escherichia coli experiments in batch were performed to validate the adapter. Fed-batch experiments with different diffusion rates and feed solutions were performed.

The adapter and the performed experiments allowed a direct comparison of the perforated ring flasks with Erlenmeyer flasks. In batch cultivations, maximum oxygen transfer capacities of 80 mmol L⁻¹ h⁻¹ were reached with perforated ring flasks, corresponding to a 3.5 times higher capacity than in Erlenmeyer flasks. Fed-batch experiments with a feed reservoir concentration of 500 g glucose L⁻¹ were successfully conducted. Based on the oxygen transfer rate, an ammonium limitation could be observed. By adding 40 g ammonium sulfate L⁻¹ to the feed reservoir, the limitation could be prevented.

The membrane-based feeding, an online monitoring technique, and the perforated ring flask were successfully combined and offer a new and promising tool for screening and process development in biotechnology.