Bioprocess Monitoring and Control in Pseudomonas cepacia and recombinant Escherichia coli cultivations

The control of cultivation conditions and its influence on growth and production have been investigated. The use of fed-batch processes have been demonstrated for several cultivation systems and so has the use of different control signals in these systems. Fed-batch cultivation of Pseudomonas cepacia with on-line measurement and control of the growth rate limiting (toxic) substrate, sodium-salicylate, was investigated. The use of an external filter unit made it possible to determine the substrate level on-line, in the bioreactor. The measured signal was used in a control-loop for manipulating the feed-flow rate around a pre-determined feed profile. Conventional analytical process equipment (dissolved oxygen sensor) have been used in a novel way to gain information on the metabolic status of Escherichia coli cells in glucose limited fed-batch cultivations. Models were constructed and compared to experimental results. It was possible to detect acetic acid formation from responses in dissolved oxygen signal upon transient pulses in glucose feed rate. The influence of medium composition have been investigated in recombinant Escherichia coli protein production (xylanase deletion derivatives from Rhodothermus marinus). Using an exponential increase in feed-flow rate, resulting in a specific growth rate below µmax, the substrate level (glucose) could be kept at a low level in the bioreactor. The use of controlled cultivation conditions have made it possible to use lactose (an inexpensive alternative to IPTG) to induce the T7lac promoter for recombinant protein production. The impact of recombinant protein production (protein L), using the temperature induced lambdaPR promotor, on growth rate and viability, was investigated and compared to the response of a non recombinant strain. Monitoring of acetic acid using flow injection analysis was carried out. The stability of the immobilised enzymes, acetate kinase and pyruvate kinase, was very low when monitoring in cultivation systems. The presence of pyruvate in the medium interfered in the analytical system. That was solved by simultaneously measuring pyruvate and subtracting its contribution from the measured acetate signal. An integrated analytical system for on-line measurements of intracellular compounds was developed. It allows measurement in cell debris containing samples by use of expanded beds for enzyme immobilisation in the analytical system