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.

BackgroundThe initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. For a successful scale-up, the operation mode on screening and production scale must be as close as possible. To enable screening under fed-batch conditions, the membrane-based fed-batch shake flask was developed. It is a shake flask mounted with a central feed reservoir with an integrated rotating membrane tip for a controlled substrate release. Building on the previously provided proof of principle for this tool, this work extends its application by constructive modifications and improved methodology to ensure reproducible performance.ResultsThe previously limited operation window was expanded by a systematic analysis of reservoir set-up variations for cultivations with the fast-growing organism Escherichia coli. Modifying the initial glucose concentration in the reservoir as well as interchanging the built-in membrane, resulted in glucose release rates and oxygen transfer rate levels during the fed-batch phase varying up to a factor of five. The range of utilizable membranes was extended from dialysis membranes to porous microfiltration membranes with the design of an appropriate membrane tip. The alteration of the membrane area, molecular weight cut-off and liquid volume in the reservoir offered additional parameters to fine-tune the duration of the initial batch phase, the oxygen transfer rate level of the fed-batch phase and the duration of feeding. It was shown that a homogeneous composition of the reservoir without a concentration gradient is ensured up to an initial glucose concentration of 750 g/L. Finally, the experimental validity of fed-batch shake flask cultivations was verified with comparable results obtained in a parallel fed-batch cultivation in a laboratory-scale stirred tank reactor.ConclusionsThe membrane-based fed-batch shake flask is a reliable tool for small-scale screening under fed-batch conditions filling the gap between microtiter plates and scaled-down stirred tank reactors. The implemented reservoir system offers various set-up possibilities, which provide a wide range of process settings for diverse biological systems. As a screening tool, it accurately reflects the cultivation conditions in a fed-batch stirred tank reactor and enables a more efficient bioprocess development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0741-6) contains supplementary material, which is available to authorized users.