PhD Thesis

Feeding Strategies Based on Probing Control for E. coli and V. cholerae Cultivations

Lena de Maré


The recombinant DNA techniques have made it possible to produce many different proteins for a wide range of applications. The vector encoding for the recombinant protein is often inserted in the bacterium \textit{E. coli} as it is a well studied and a well-known organism. To achieve a high productivity, it is important to reach a high cell density. This is obtained through fed-batch operation where the nutrient, usually glucose, is added continuously. The feeding strategy should be designed to avoid starvation and overfeeding. This is not an easy task as on-line measurements of key variables are normally not available. A probing feeding strategy using the measurements of the standard dissolved oxygen sensor is developed by M. Åkesson. The key idea is to superimpose pulses on the feed-rate and make use of the responses in the dissolved oxygen in a feedback algorithm. Also, when the maximum transfer capacity of the reactor is reached the feed-rate is decreased in order to keep the reactor working under aerobic conditions. The strategy has been successfully implemented in cultivations with different \textit{E. coli} strains and on different scales. The probing feeding strategy is further developed in many aspects in this thesis. Firstly, it is implemented with good results also in \textit{V. cholerae} cultivations, which shows how general the strategy is. Also, a model of a bio-reactor operating in fed-batch mode is presented and verified. The effect on the tuning rules of the probing controller is investigated. The probing feeding strategy is further improved for a more efficient product synthesis. This new fermentation technique manipulates the temperature when the maximum oxygen transfer capacity of the reactor is reached. The strategy consists of a mid-ranging controller structure and a modified probing controller. It is analysed and evaluated in experiments and simulations. Furthermore, some \textit{E. coli} production strains need additions of amino acids or complex media besides the carbon nutrient to grow and produce the recombinant protein. The probing control concept is therefore extended in order to handle these situations. Feeding strategies for dual feeding of amino acids or complex media and glucose are developed.


Probing control, Fed-batch bioreactors, Feeding strategy, Parameter estimation, Complex media, Amino acid, Mid-ranging control, Temperature

PhD Thesis ISRN LUTFD2/TFRT--1076--SE, Department of Automatic Control, Lund University, Sweden, June 2006.

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