Construction Of Recombinant Saccharomyces Cerevisiae Strains For Improved Ethanol Yield And The Cleaner Fermentation Process Study

Demand for ethanol, as a substitute for gasoline, is expected to increase because of concerns related to national security, economic stability, environmental impact, and global warming. Saccharomyces cerevisiae was used extensively in the ethanol fermentation.In this investigation we employed the molecular biology techniques to construct recombinant strains of the yeast Saccharomyces cerevisiae, in which the GDH1 gene, encoding the glutamate dehydrogenase were disrupted to direct the ammonium metabolic flux to the pathway consuming NADH. The fermentation experiment results showed the ethanol yield and acetic acid concentration increased and the glycerol concentration in fermentation broth discreased. This means that the ammonium metabolisim consumed more NADH.A diploid strain KZ4-D (Mat a/αURA3- fps1Δ::repeat gpd2Δ::repeat GLT1-PGK1) was constructed through renewing the marker gene and crossing, and then chosen as the study object owing to the more ethanol yield. We investigate the growth and metabolic of KZ4-D in various fermentation conditions and the results indicated that the KZ4-D growth less slowly but the ethanol yield was 3.1% higher compared to the wide type strain CK-D.In this work, we studied the process in which clarified corn liquid was used as the fermentation substrate. The data from lab and from industry scale showed, comparing the corn mash fermentation, there are many advantages in the clarified corn liquid fermentation process, such as the pipeline block had been reduced significantly, the operation was more stable, and the most important point was the ethanol yield and the ethanol productivity was increased 2.56% and 28%, respectively.A simple model was proposed using the logistic equation for growth, the Luedcking-Piret equation for ethanol production and Luedeking-Piret-like equations for reducing sugar consumption. The model appeared to provide a reasonable description for each parameter during the growth phase. Moreover we optimized the fermentation conditions and the medium composition using statistical experimental design to increase the ethanol yield in the clarified corn liquid fermentation process. And the optimal contitions for ethanol yield were found: (NH4)2HPO4 1.094g/L,(NH4)2SO4 0.2 g/L,MgSO4·7H2O,KH2PO4 0.5 g/L,CaCl2·2H2O 0.01 g/L,temperature 33.6℃,initial pH 5.5,shake rate 110r/min. The ethanol yield was increased 1.83% under the optimal conditions.