Probing Control of Glucose Feeding in Escherichia coli Cultivations

Abstract: Production of many proteins can today be made using genetically modified organisms. One of the most frequently used host organisms is the bacterium Escherichia coli. A difficulty encountered in cultivations of E. coli is the accumulation of the metabolic by-product acetate which inhibits cell growth and production of a desired protein. Formation of acetate occurs under anaerobic conditions but also in situations with excess of the carbon/energy source that usually is glucose. In fed-batch processes the glucose feed rate can be manipulated to avoid acetate formation, but most feeding strategies require considerable process knowledge to handle process variations. On-line measurements for the relevant process variables are far from being standard which complicates the realization of strategies based on feedback control. This thesis presents a glucose feeding strategy for E. coli cultivations that avoids acetate formation in spite of process variations and without prior knowledge of the particular strain and product. The key idea is to exploit a characteristic saturation in the cellular respiration at the onset of acetate formation. By superimposing short pulses in the glucose feed rate, on-line detection of acetate formation can be made using a standard dissolved oxygen sensor. This information is used in a feedback algorithm that adjusts the feed rate to avoid acetate formation while maintaining a high glucose supply. The feed rate is also restricted to ensure aerobic conditions when the maximum oxygen transfer capacity to the culture is reached. The feasibility is demonstrated by simulations as well as laboratory-scale experiments with several E. coli strains under various operating conditions. Tuning rules that assume a minimum of process specific information are derived and a stability analysis is given. The feeding strategy relies on good control of the dissolved oxygen concentration. Variations in the oxygen dynamics during a fed-batch cultivation often cause tuning problems when using a controller with fixed parameters. A control approach based on gain scheduling from the stirrer speed is suggested.