# Diffusion-driven fed-batch fermentation in perforated ring flasks

## TL;DR

A new flask design with a perforated ring and membrane-based feeding improves oxygen transfer and monitoring for bioprocess development.

## Contribution

A perforated ring flask with integrated oxygen transfer monitoring is introduced for efficient bioprocess screening.

## Key findings

- Perforated ring flasks achieved 80 mmol L−1 h−1 oxygen transfer, 3.5 times higher than Erlenmeyer flasks.
- Fed-batch experiments with 500 g glucose L−1 were successfully conducted using the new flask design.
- Ammonium limitation was observed and resolved by adding 40 g ammonium sulfate L−1 to the feed reservoir.

## Abstract

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.

The online version contains supplementary material available at 10.1007/s10529-024-03493-0.