# Foam Formation in Shake Flasks and Its Consequences

## TL;DR

This paper shows that foaming in shake flasks can significantly increase oxygen transfer and affect experiment reproducibility.

## Contribution

The study reveals that foaming in non-baffled shake flasks can triple oxygen transfer capacity and impact reproducibility.

## Key findings

- Foaming in shake flasks can increase the maximum oxygen transfer capacity (OTRmax) by up to threefold.
- Foaming can be caused by baffles, sensor spots, or the out-of-phase phenomenon in non-baffled flasks.
- Foaming affects reproducibility and process conditions, especially in high-viscosity microbial cultures.

## Abstract

Foam formation in stirred tank fermentation processes is a well‐studied phenomenon. However, foaming in shake flask cultivations is rarely considered. Non‐baffled shake flasks, in particular, are generally considered to prevent foaming problems. However, under certain process conditions, foaming in non‐baffled shake flasks can occur. In this study, phenomena of foam formation in shake flasks, their impact on the maximum oxygen transfer capacity (OTRmax), and experimental reproducibility are investigated. It is shown that foaming events in shake flasks can increase the OTRmax by up to threefold. This enhanced OTRmax alters process conditions and, thereby, affects the reproducibility of experiments. Foaming in shake flasks can be induced by elements such as conventional baffles or sensor spots that are used for online measurement. Moreover, a connection between the out‐of‐phase phenomenon and foam formation was discovered in non‐baffled shake flasks. This is especially important when cultivating microorganisms at elevated viscosities. Hence, foaming in shake flasks should be considered as significantly altering process conditions, compared to non‐foaming cultures. Ensuring in‐phase cultivation conditions and unhindered liquid flow in shake flasks may help to avoid foaming.

Practical application: This work provides insights into foam formation in non‐baffled shake flasks and its resulting implications. Foaming can increase the maximum oxygen transfer capacity and, thus, affect process conditions. The reproducibility can be severely reduced, and a comparison between foaming and non‐foaming cultivations is only possible to a limited extent. Foaming can be induced by baffles or internals, such as small sensor spots, used for online monitoring. Additionally, foaming can be caused by the out‐of‐phase phenomenon. This is of particular importance when cultivating microorganisms at elevated viscosities. This paper is intended to raise awareness of the topic of foam formation in the shake flask and help to correctly interpret this phenomenon.