Abstract
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.
Highlights
The initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions
The fed-batch operation mode is chosen for biological systems that evoke challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism when exposed to high initial substrate concentrations
The Reichelt (1) dialysis membrane used in the following experimental study is permeable to low molecular weight solutes as glucose and ions but impermeable to high molecular weight proteins [30]
Summary
The initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. The fed-batch operation mode is chosen for biological systems that evoke challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism when exposed to high initial substrate concentrations. This cultivation strategy has proven to be advantageous for a variety of microorganisms such as Escherichia coli [5], Saccharomyces cerevisiae [6], Bacillus subtilis [7] and Pichia pastoris [8]. The availability of fed-batch screening tools is an essential prerequisite for a fast-track implementation of production processes
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