Analysis and Control of Continuous Microaerobic Ethanol Production by Yeast

Doctoral thesis, 1997

Glucose fermentation by Saccharomyces cerevisiae, and xylose fermentation by Candida utilis and Pichia stipitis, were investigated under microaerobic conditions. A dynamic experimental method named oxygen programmed fermentation (OPF), and a method for microaerobic RQ control in continuous culture have been developed. An OPF allows a relatively fast identification of the range of interesting oxygen uptake rates, and gives information on the dynamics of the culture in response to a slow washout of oxygen. Results obtained with this method were in good agreement with the characteristics of the three yeasts. In situ NAD(P)H fluorescence measurements during OPF:s with S. cerevisiae indicated, that the intracellular redox level increased with decreasing oxygen concentration up to a certain level, at which glycerol was produced as a redox sink. Different glycerol-3-phospate dehydrogenase deletion mutants of S. cerevisiae responded remarkably similar in OPF experiments. Under microaerobic conditions, the .DELTA.gpd1 / .DELTA.gpd2 double mutant produced somewhat more ethanol and only traces of glycerol. In microaerobic, nitrogen-limited chemostat cultures of S. cerevisiae, the ethanol yield was up to 8 % higher compared to anaerobic, carbon-limited conditions, depending on the dilution rate. Apart from NADH reoxidation, the major effect of the oxygen was to increase the magnitude of the nitrogen limitation. The respiratory qotient (RQ) was controlled in continuous cultures of S. cerevisiae over a range of setpoints of 6 to 80, by changing the inlet gas composition with a PID controller with gain scheduling. The ethanol yield reached a flat maximum at RQ values between 12.5 and 50. The decrease in the glycerol yield at lower RQ was accompanied by an increase in the biomass yield. Metabolic flux analysis indicated that the disappearance of glycerol coincided with the cyclic operation of the TCA cycle reactions. This means, that glucose is not catabolized oxidatively until there is sufficient oxygen to allow for redox balancing of the produced NADH. A combination of anabolic limitation, and a controlled oxygen addition, should therefore have a large potential for increasing the ethanol yield.