Isolation And Identification Of Thermotolerant Yeast And Basic Characteristics Of Ethanol Fermentation By Protoplast Fusion Strain

Thermotolerant ethanol-producing yeast can be selectively bred for the industrial production of fuel ethanol. This method can improve ethanol productivity and reduce the cost of fermentation and distillation while mitigating the pollution of fermentation waste.In this study,59strains of thermotolerant yeast were isolated from samples of different sources and2strains of thermotolerant ethanol-producing yeast WXT3and K213were isolated through temperature gradient screening and TTC rescreening. The isolated strains WXT3and K213grew at25-48℃or25-45℃, respectively and could adapt to wide pH range (2.0-9.0,3.0-9.0). The growth and proliferation of the two strains did not require vitamins and other growth factors and they could use several carbon sources including five-carbon sugar. The two strains could tolerate ethanol concentration of6%(v/v) and glucose concentration of40%(w/v). WXT3was able to tolerate NaCl concentration of10%(w/v) and both strains could grow on culture medium containing0.01%cycloheximide. They produced acetic acid but not amyloid compounds. According to nucleotide sequence of5.8S+ITS region, morphology and physiological characteristics, WXT3and K213were respectively identified as Ogataea sp. and Kluyveromyces marxianus and named as Ogataea sp. WXT3and Kluyveromyces marxianus K213.The analysis of ethanol fermentation characteristics of isolated strains showed that44.57g/L ethanol (0.30g/g glucose) was produced through fermentation of15%(w/v) glucose by WXT3at42℃for96hours; the conversion rate was58.3%of the theoretical value;68.77g/L ethanol (0.46g/g glucose) was produced through fermentation of15%(w/v)glucose by K213at42℃for48hours; the conversion rate was89.9%of the theoretical value.To obtain yeast strains capable of producing high-concentration ethanol through fermentation at high temperature, the isolated thermotolerant strain K213and industrial angel yeast were selected as parent strains. Using protoplast fusion, fermentation strains with good performance were bred and the characteristics of ethanol fermentation were researched. Firstly, using double inactivated protoplast as genetic marker, protoplast fusion was performed for K213and Angle yeast with PEG, to obtain thermotolerant fusant Ka911and Ka914, which had high ethanol productivity. Fusant Ka911and Ka914possessed stable hereditary traits were subcultured for five generations (once every24hours). Compared to the first generation, no significant changes were observed in terms of growth and ethanol productivity. The cell volume, colony size and morphology of fusant Ka911and Ka914were similar to those of parent strain K213. Sequence analysis of5.8S+ITS rDNA showed that there was significant difference between the bases of fusant strain and Angel yeast strain; the difference was observed only at several loci in K213.Next, the fermentation characteristics of fusion strains were investigated. Glucose fermentation at45℃by fusion strains Ka911and Ka914produced38.15g/L and42.84g/L ethanol respectively, which was22.3and25.1times of that (1.71g/L) by Angel yeast strain at the same fermentation temperature; or87.8%and98.6%of that (43.46g/L) by thermotolerant parent strain Kluyveromyces marxianus K213. At48℃,42.35g/L and38.00g/L ethanol was respectively produced, which was22.4and20.1times of that (1.89g/L) by parent strain Kluyveromyces marxianus K213. The fermentation temperature of fusion strain was increased by respectively6℃and3℃, compared Angle yeast strain and K213; ethanol production was similar to that of parent strains. By protoplast fusion, yeast strains capable of higher ethanol productivity at higher temperature were successfully obtained.