Biochemical and Mathematical Modeling of Microaerobic Continuous Ethanol Production by Saccharomyces cerevisiae

Citation

Grosz, Ron (1987) Biochemical and Mathematical Modeling of Microaerobic Continuous Ethanol Production by Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/29dc-ay81. https://resolver.caltech.edu/CaltechETD:etd-03192008-120209

Abstract

The effect of the aeration intensity in the vicinity of 1Oppb of dissolved oxygen upon the steady state, continuous ethanol production by Saccharomyces cerevisiae is explored. The specific ethanol productivity increased by 30 to 50% as the aeration rate was reduced, but decreased to the original level as the aeration was further reduced to the lowest rates. These metabolic changes occurred when the respiration rate contributed negligibly to ATP energy production, excluding the Pasteur mechanism, and when the residual glucose level saturated the glucose transporter, excluding glucose kinetics as the cause.

To expose the mechanism of the metabolic changes, the intracellular concentrations of ethanol, glycerol, ATP, glucose 6-phosphate, pyruvate, and NADH, the activities of the hexokinase, alcohol dehydrogenase, fumarase, and isocitrate dehydrogenase, and the cell viability were assayed. Rate-limiting steps were identified by the accumulation of upstream and the depletion of downstream metabolites. The enzyme activity and cofactor measurements elucidated the causes of the rate limitation.

Based on the assays, the metabolic acceleration with decreasing aeration was the result of an increasing glucose transporter activity, and ATP was the most likely activator. The reversal at yet lower aerations resulted from the continued accumulation of ATP until the downstream glycolytic kinases were inhibited.

High concentrations of silicone polymer antifoam decreased the resistance to glycerol transport across the cell membrane, enhancing glycerol production at the expense of ethanol production. Such cultures attained lower ATP concentrations.

The biomass concentration in the fermentor was occasionally found to undergo hysteresis, characterized by extinction and ignition as critical aeration rates were passed. Hysteresis and associated phenomena were prevented by the addition of yeast extract and the removal of the antifoam from the medium. The higher ATP concentration of the low antifoam culture and the biosynthetic intermediates of yeast extract must participate in this transition.

Mathematical models account for all observed phenomena. Featured by the model are the ATP activation of the glucose transporter, ATP wastage by ATPases and such processes, and oxygen induction of ATP waste reactions. Singularity theory was invoked to account for the transition from the hysteresis to monotonic biomass versus aeration bifurcation diagrams.

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