Polymer-based controlled-release fed-batch microtiter plate – diminishing the gap between early process development and production conditions

Abstract

Background Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. Results Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. Conclusions The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible. Electronic supplementary material The online version of this article (10.1186/s13036-019-0147-6) contains supplementary material, which is available to authorized users.

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

“…Recent developments also enable fed‐batch conditions in MTPs. Two typically applied systems are the enzymatic degradation of polysaccharides and polymer‐based glucose release from a silicone elastomer matrix . These techniques provide improved scalability of bioprocesses from MTPs to industrial‐scale production .…”

“…These techniques provide improved scalability of bioprocesses from MTPs to industrial‐scale production . Furthermore, the effect of different glucose release rates can be investigated at an early stage of process development . However, all of these studies focused on the application of fed‐batch conditions in the expression, or main culture.…”

Glucose‐containing polymer rings enable fed‐batch operation in microtiter plates with parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH

“…The presented microtiter plate uses the polymer‐based controlled‐release system to introduce fed‐batch conditions. Glucose is released continuously from the polymer matrix and was described to be stable and reproducible (Keil et al, ). However, due to the manual manufacturing of the polymer rings, the cut surface of the polymer ring caused a strong and abrupt initial glucose release.…”

“…Amylases and proteases do not impact glucose release characteristics. Furthermore, glucose release was shown to be highly reproducible and less sensitive against changing cultivation conditions compared to the enzymatic release system (Keil, Dittrich, Lattermann, Habicher, & Büchs, ). Nevertheless, the enzymatic glucose release system is advantageous to the current polymer‐based release system in terms of online monitoring.…”

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

“…Recently, Keil et al. provided a detailed characterization of glucose release kinetics with varying media conditions and temperatures . When compared to the enzymatic release system, the polymer‐based release system showed to be less sensitive against moderate changes of pH and temperature …”