The application of a novel sampling device to the on-line analysis of fermentation broth
This thesis describes the development of an automated, aseptic, sampling, centrifugation and analysis system for fermentation broth, and its link to a process control system for the control of sugars and organic acids in fermentation processes. Experiments were done to determine the separation efficiency of the microcentnfuge on suspensions of E. coli and S. cerevisiae cells. Reproducibility tests showed the system to be suited to on-line use, and on-line HPLC results compared well with off-line enzymatic assays of glucose and acetate for samples taken during an E. coli K12 fermentation. The on-line monitoring system was linked to a control system developed by another researcher to enable on-line HPLC results to be used to control a fermentation process. The thesis presents two examples of the use of the combined system in controlling acetate build-up in a fed-batch fermentation. Operating fed-batch fermentations provided suitable conditions for the testing of the on-line monitoring system in controlling the build-up of galactose and acetate in the fermenter. Experiments looked at the effects of various fermentation conditions on the expression of α-amylase produced in a recombinant E. coli, JM1O7 + pQR126, and secreted into the periplasm. Fed-batch fermentations were performed at different growth rates and feed profiles. Further physiological studies were then performed using continuous culture techniques to look at the effect of acetate concentration on the maximum specific growth rate and α-amylase production; and the effect of dilution rate on the overflow of substrate to acetate and production of α-amylase. Results show that high and low growth rates inhibit α-amylase production, as do high acetate and galactose concentrations. High acetate concentrations also reduce the maximum specific growth rate. These experiments highlight the need for monitoring and controlling galactose and acetate concentrations in fermentation processes.