The development of on-line, sensor-based control systems capable of directly monitoring and responding to biochemical events inside a cell would be a major advance in the regulation of the complex biochemical reactions needed to synthesize commercial products. Using the butanol fermentation as a model system in Phase I, the feasibility for process control was demonstrated by running continuous fermentations in which the reducing agent, benzyl viologen, was successfully used to control culture fluorescence at a predetermined set point. It is the premise of the Phase II project that using additives to control the reduction state of the cell is a generic phenomenon that is likely to result in novel operating strategies for a broad range of fermentation and cell cultivation processes. The research program is designed to study the relationship between the intracellular reduction state as measured by the ratio of ?NAD(P)H/(NAD(P) + NAD(P)H! and enhanced product formation in several broad types of processes. Building upon the results of Phase I, the butanol fermentation will be employed as the system to quantify product formation as a function of the reduction state in anaerobic procaryotes. Recombinant E. coli will be used to study aerobic procaryotic cells to determine the relationship between the reduction state and the optimal expression of genetically engineered polypeptides and proteins. Eucaryotic cells will be studied by using hybridomas as the model system to examine the production of monoclonal antibodies as a function of cell reduction state. In all three systems, the concentration of intracellular NAD(P)H will be used as the control variable for regulating product biosynthesis. Sophisticated systems for monitoring and controlling fermentation and mammalian cell cultivation processes are needed for the scale-up and commercial production of many products of biotechnology, such as plasminogen activator, monoclonal antibodies, interfon, and interleucon-2. The currently available on-line process sensors are few in number (i.e. temperature, pH, dissolved oxygen, redox, etc.) and limited by the fact that they measure environmental variables and not the intracellular characteristics. None of the currently available on-line process sensors are able to measure intracellular metabolism directly. The development of on-line process sensors are able to measure intracellular metabolism directly. The development of on-line, sensor-based control systems capable of directly monitoring and responding to biochemical events inside a cell would be a major advance in the regulation of the complex biochemical reactions needed to synthesize commercial products. The objective for the research is to use the recently developed instrumentation for monitoring the health of cells. This ability to measure changes directly in intracellular metabolism will be the motivation for developing innovative process control techniques. It is anticipated that the proposed research will ultimately result in -2- improved process control with increased productivity and lower costs for a broad range of commercial fermentation and cell cultivation processes.
