Fed-Batch-Technik mit Zeit verzögertem Einsetzen der Nährstoffzufuhr

Abstract:
The production of microbial products needs an optimum microorganism for each process. To select the best one requires a lot of experimental work. Due to high cost and time efforts these screening experiments are normally carried out in batch mode with parallel run small scale cultures. However, industrial production processes often use the fed-batch process to overcome the deficiencies of yield decreasing phenomena. The different ways of running screening and production processes result in quantitatively and qualitatively different metabolism activity, which may lead to a wrong selection of microorganism. There is a need for a cost efficient and easy to handle fed-batch system for the primary screening. The newly developed and tested fed-batch shake flask with a diffusion driven release of nutrients from a reservoir should be optimized for this application. Focus was put on the delayed release of nutrient to avoid an excess of substrate at the beginning of cultivation. Building in small cigarette filters into the diffusion tip was a first step to achieve an additional retarded release of nutrient. The use of transparent material for constructing the fed-batch system makes the process steps more evident and easier to observe. Mistakes and problems influencing the release of nutrient can easily be seen. The new developed vacuum filling process is minimizing problems of handling of the fed-batch system. Its optimization results in a more efficient and better adapted system to fulfill the requirements of the high-throughput screening. A better understanding of the release steps and material flow of nutrient was worked out with the newly developed transparent diffusion tip. Also osmotic based penetration of water from the flask medium into the reservoir and its influence on the concentration driven release of nutrient was investigated for the first time. Now, there is more information available on the quite complex process, which might be used in the future for a modelling of the process. Several experiments have been conducted with the high yield Lysine producing strain C. glutamicum DM 1730 using an optimized mineral medium by adding urea. The results make evident that there is no limitation of oxygen even at high glucose release rates using the fed-batch cultivation contrary to batch process. Because of differences in the release of glucose the growth of bacterium is not constant under same conditions.

Keywords:
fed-batch shake flask, delayed feeding, Corynebacterium glutamicum DM 1730