Evaluierung eines membranbasierten Fed-Batch-Systems für den Schüttelkolben am Beispiel von E. coli BL21 (DE3) pRhotHi-2-Ec FbFP

Abstract:
Fed-batch is probably the most frequently used operation mode for the biotechnological production in industry. Nevertheless, the screening of a suitable production strain is performed in batch-mode. This could lead to a false choice of strain with fatal economic consequences. With a screening in fed-batch mode it would be possible to overcome this problem. Few fed-batch systems for smale scale have been realized by a diffusive release of glucose. The one used in this work releases glucose from a reservoir over a dialysis membrane into the culture broth. By continuous rotation, it is in permanent contact with the medium. For a better understanding of the diffusion processes, the glucose release into the flask was measured by a refractometer. Besides this, reverse water diffusion was detected by observation of the feed volume in the reservoir. The quantification of the two mass fluxes allows a further characterization of their influence on the glucose release. Furthermore, fermentations of the Escherichia coli strain BL21 (DE3) pRhotHi-2-EcFbFP were performed to evaluate this fed-batch-system for shake flasks. A specific attribute of this bacterial strain is the oxygen independent production of a fluorescent protein, which can be photometrically detected. A comparison of cultivation in batch and fed-batch mode showed differences in growth and protein production. At the beginning of fermentation glucose accumulates due to the maximal release. To overcome this phenomenon, the fermentation was started with a low glucose concentration. Later a higher concentrated solution was added to supply the increasing demand of nutrient and to avoid a glucose accumulation.

Keywords:
fed-batch shake flask, additional feeding, E. coli BL21 (DE3) pRhotHi-2-Ec FbFP