Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding.

An automated glucose feeding strategy that avoids acetate accumulation in cultivations of Escherichia coli is discussed. We have previously described how a probing technique makes it possible to detect and avoid overflow metabolism using a dissolved oxygen sensor. In this article these ideas are extended with a safety net that guarantees that aerobic conditions are maintained. The method is generally applicable, as no strain-specific information is needed and the only sensor required is a standard dissolved oxygen probe. It also gives the highest feed rate possible with respect to limitations from overflow metabolism and oxygen transfer, thus maximizing bioreactor productivity. The strategy was implemented on three different laboratory-scale platforms and fed-batch cultivations under different operating conditions were performed with three recombinant strains, E. coli K-12 UL635, E. coli BL21(DE3), and E. coli K-12 UL634. In spite of disturbances from antifoam and induction of recombinant protein production, the method reproducibly gave low concentrations of acetate and glucose. The ability to obtain favorable cultivation conditions independently of strain and operating conditions makes the presented strategy a useful tool, especially in situations where it is important to get good results on the first attempt.

On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients.

Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.

Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae.

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO(2) semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto-Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.