Methods in Molecular Biology (2022) 2436: 223–240

DOI 10.1007/7651_2021_424

© Springer Science+Business Media, LLC 2021

Published online: 15 September 2021

A Guideline to Set Up Cascaded Continuous Cultivation

with E. coli Bl21 (DE3)

Julian Kopp and Oliver Spadiut

Abstract

Continuous processing allows to maximize space-time yields and is implemented in many industrial

branches. However, in manufacturing of value added compounds produced with microbial hosts, continu-

ous processing is not state-of-the-art yet. This is because fluctuating productivity causes unwanted process

deviations. Cascaded continuous bioprocessing, unlike conventional continuous process modes, was found

to result in stable productivity. This manuscript serves as a guideline how to set up a cascaded continuous

cultivation with Escherichia coli BL21 DE(3).

Key words Cascaded continuous cultivation, Escherichia coli BL21(DE3), Long-term stable pro-

cesses, Microbial continuous cultivation, Stable productivity

1

Introduction

Continuous bioprocessing has been implemented in many indus-

trial branches [1]. This is because continuous production allows a

major improvement in volumetric throughput by facilitating

manufacturing in smaller production scales [1–3]. In respect to

biotechnological applications, Herbert et al. demonstrated the

economic feasibility of continuous bioprocesses over batch cultiva-

tions back in 1956 [4]. Continuous cultivation was already intro-

duced by Monod, Novick, and Szilard in the early 1950s

[5–8]. The so-called chemostat cultivation was originally developed

to investigate cell physiology allowing prediction of environmental

conditions on host cells [7–9]. Biotechnological approaches in

industry nowadays try to follow the principles of Herbert et al.

[4], to boost the space-time yield of conventional processes. Unfor-

tunately, this approach proved to be more tricky than expected

[10]. Many recombinantly produced components are toxic for

host cells at required industrial product titers [11], potentially

causing process deviations. As host cells try to escape the formation

of the target product, effects might lead to subpopulation diversifi-

cation [11–13]. The formation of subpopulations (either pheno-

typic or genotypic) [14–16] leads to unwanted process deviations.

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