Screening A Thermotolerant Yeast Mutant With UV And DES-induced Mutagenesis And Its Application To VHG Ethanol Fermentation

Employing thermotolerant yeasts in ethanol fermentation has received much attention due to many benefits such as its lower production cost. But there are challenging problems needed to tackle before ethanol fermentation can be successfully performed at high temperature. The most difficult problems is cell aging and death resulting from high fermentative temperature. As a result, how to enhance yeast activity under high temperature is critically important. To this end, the research was fouced on screening the mutants with good thermolerance, and both optimizing fermentative conditions and understanding alterations in physiological and biochemical aspects of the mutants were emphasized in this study.Firstly, the experiments were performed with the purpose of screening variants which can produce maximal ethanol at high temperature. The parental strain was treated with UV-irradiation and diethyl sulfate (DES), singly and in combination. Through repeated isolations, mutants UD1 and UD4 were finally identified as the best two variants with the highest temperature and glucose tolerance.Then, the research was carried out to optimize the fermentative conditions by uniform design approach . An optimal combination of variables was eventually established : 330 g.L^-1 glucose, 1.1 mmol.L^-1 Ca²⁺, 4.0 mmol.L^-1 Mg²⁺, 0.5 mmol.L^-1 K+, 8.2×10⁷ cells.mL^-1 inoculum. Under such conditions, 16.7%(v/v)of final ethanol in fermentative broth was obtained, representing about 34% increase compared with non-optimization conditions, with glucose utilization increased by 24.5%.The research was further condcted in a stirred tank fermentor (5L) based on the above optimization results. The experimental data showed that the mutant UD1 has strong fermentative ability and high cell viability throughout the fermentative process. Meanwhile, the fermentative time was reduced from 65 to 48 h, and the final ethanol concentration achieved 16.8% (v/v). But the amount of glycerol generated by the mutant UD1 was higher compared with non-optimization conditions possibly due to the fact that a protective effect of glycerol is benificial to yeast under stress conditions.Finally, the research was performed to understand the physiological and biochemical aspects associated with the mutant UD1 through analyses of the plasma membrane ATPase, fatty acid composition and membrane permeability. It suggested that a higher plasma membrane ATPase activity and a lower membrane permeability were related to the increased stress tolerance of mutant UD1. Moreover, the content of membrane fatty acid of the mutant UD1 was 86.66% in the total extraction, while the corresponding value in the parental strain was only 42.28% . Through analyses by GC-MS technique, the six kinds of fatty acid were identified, while ketones were largely found in the fusant SPSC. These data may assist in understanding a relationship between yeast thermotolerance and its plasma membrance.