Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

Abstract

BackgroundScreening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli.ResultsIn combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation.ConclusionsThe newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0917-8) contains supplementary material, which is available to authorized users.

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane‐based fed‐batch shake flasks

“…With the assumption of a constant glucose release rate, the length of the batch phase depends on the glucose consumption rate, which depends on the initial biomass concentration and the specific growth rate (Huber et al, ). To prevent extended batch phases with possible oxygen limitations (Philip et al, ), an appropriately high initial biomass concentration was chosen. Due to growth, the initial glucose consumption rate increases further and as soon as the consumption rate is higher than the release rate, the accumulated glucose decreases (Bähr et al, ; Huber et al, ; Jeude et al, ; Philip et al, ).…”

“…Once nitrogen is depleted, growth stops and B. licheniformis uses the available glucose for cell maintenance. Hence, the stoichiometric relations change resulting in an increased demand of oxygen per consumed amount of glucose (Philip et al, ). This example shows that changing cultivation conditions have to be considered when using the oxygen‐based yield coefficient.…”

“…the stoichiometric relations change resulting in an increased demand of oxygen per consumed amount of glucose (Philip et al, 2018). This example shows that changing cultivation conditions have to be considered when using the oxygen-based yield coefficient.…”

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

“…It is commonly known that lower osmolality is beneficial for the growth of E. coli. Due to lower initial glucose concentrations in fed‐batch media, the media have lower osmolality and are therefore more favorable for growth.…”

A model‐based framework for parallel scale‐down fed‐batch cultivations in mini‐bioreactors for accelerated phenotyping

“…An initial challenge of these systems was the difficulty in implementation of controlled feeding strategies, as in laboratory bioreactors. However, various solutions have been developed recently to enable fed-batch-like conditions, such as the gradual supply of glucose to the culture through enzyme-based glucose release systems (Krause, Neubauer, & Neubauer, 2016), the application of micropumps for continuous feed supply (Gebhardt, Hortsch, Kaufmann, Arnold, & Weuster-Botz, 2011), model-based intermittent feeding (Sawatzki et al, 2018), and membrane-based substrate release systems (Philip et al, 2018). These improvements bring the screening system closer to actual cultivation conditions, such that a better performing strain can be selected for scale-up.…”