【作者】 许晓菁；

【导师】 马平生；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 能源匮乏的日益加剧和温室效应对全球气候造成的影响,使得人们越来越关注利用生物质材料生产替代能源乙醇的研究。利用酿酒酵母发酵生产乙醇是当前应用非常广泛的乙醇生产方法。为了获得乙醇产量提高的菌株,本文作者在本实验室他人工作的基础上,进一步通过分子生物学手段构建基因工程菌株。通过比较,挑选出性能优良的KZ4-D菌株进行发酵性能的考察以及工艺条件的摸索。为了进一步引导酿酒酵母中的铵代谢流向利用NADH的谷氨酸途径,本论文中将原有工程菌株中的GDH1基因进行缺失,并通过交配和等位基因分离等手段获得一系列GDH1基因缺失的工程菌株。通过对这些菌株生长代谢情况的考查我们发现,已经过量表达GLT1基因的菌株中,GDH1基因缺失后,甘油产量降低,乙醇产量提高,乙酸产量有所提高,说明铵代谢进一步消耗了NADH。为了得到适于工业生产的基因工程菌株,我们在对现有菌株进行比较分析后选择KZ4 (Mat a URA3- fps1Δ::repeat gpd2Δ::repeat GLT1-PGK1)作为重点考察对象。将KZ4菌株通过标记基因恢复和交配手段得到双倍体菌株KZ4-D。当接种量控制为初始OD660为2.0时,可以满足KZ4-D菌株的发酵要求,其乙醇产量比野生型菌株CK-D提高了3.1%。本文中研究了利用玉米糖化清液进行发酵的工艺。实验数据显示,利用玉米清液发酵,比传统的带渣发酵具有更多优势。例如,酶的用量降低,生产更稳定,管路阻塞减少,最重要的是乙醇产量和单位体积的生产能力分别提高了2.56%和28%。在一系列实验数据的基础上,我们建立了清液发酵过程的动力学模型,用logistic方程对菌体生长进行拟合,用Luedcking-Piret方程对乙醇形成进行拟合,用Luedeking-Piret-like方程对基质消耗进行模拟,都取得了较好的效果。为了进一步提高乙醇产量,我们利用统计学方法对清液发酵条件进行优化处理,确定了摇瓶实验中清液发酵的最优化条件为:(NH4)2HPO4 1.094g/L,(NH4)2SO4 0.2 g/L,MgSO4·7H2O,KH2PO4 0.5 g/L,CaCl2·2H2O 0.01 g/L,温度为33.6℃,初始pH5.0,转速为110r/min。经优化后,KZ4-D的乙醇产量比优化前提高了1.83%。更多还原

【Abstract】 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.更多还原