Fed-batch operation in special microtiter plates: a new method for screening under production conditions

Batch and fed-batch operation result in completely different physiological conditions for cultivated microorganisms or cells. To close the gap between screening, which is hitherto exclusively performed in batch mode, and fed-batch production processes, a special microtiter plate was developed that allows screening in fed-batch mode. The fed-batch microtiter plate (FB-MTP) enables 44 parallel fed-batch experiments at small scale. A small channel filled with a hydrogel connects a reservoir well with a culture well. The nutrient compound diffuses from the reservoir well through the hydrogel into the culture well. Hence, the feed rate can easily be adjusted to the needs of the cultured microorganisms by changing the geometry of the hydrogel channel and the driving concentration gradient. Any desired compound including liquid nutrients like glycerol can be fed to the culture. In combination with an optical measuring device (BioLector), online monitoring of these 44 fed-batch cultures is possible. Two Escherichia coli strains and a Hansenula polymorpha strain were successfully cultivated in the new FB-MTP. As a positive impact of the fed-batch mode on the used strains, a fourfold increase in product formation was observed for E. coli. For H. polymorpha, the use of fed-batch mode resulted in a strong increase in product formation, whereas no measurable product formation was observed in batch mode. In conclusion, the newly developed fed-batch microtiter plate is a versatile, easy-to-use, disposable system to perform fed-batch cultivations at small scale. Screening cultures in high-throughput under online monitoring are possible similar to cultivations under production conditions.