| Fermentation production of fuel ethanol is attracting increasing attention.Currently,yeast strains were mostly used in the production of industrial ethanol fermentation.The main goal of this research was to find a Saccharomyces cerevisiae strain which showed high ethanol production with heat and ethanol resistance by means of mutation,domestication and protoplast fusion.Firstly,rice wine yeast M cultured at 30℃and Angel yeast A were chosen as the initial strains.Strain M-UV-DES 3(abbreviation M-3) growing at 37℃was screened through combination mutation of UV and DES.The ethanol yield was 0.475 g·g-1,which increased 10.9%in comparison with the original strain M.TTC method was used in screening of Angel yeast A.The ethanol yield of most strains has achieved over 0.41g·g-1.Ethanol production from A-8 was obviously improved to 72.0 g·l-1 in 37℃fermentation and its ethanol was 0.481g·g-1.Secondly,the effects of glucose concentration to M-3 and A-8 fermentation were studied.The fermentation performances of yeasts were simultaneously inhibited by both ethanol and glucose,where initial glucose concentration above 200g.l-1.The maximum of ethanol maintained at a similar level.In this case,ethanol gradient domestication was applied to improve their alcohol-resistant properties.Through repeated batch domestications,the ethanol tolerance capacity of both M-3 and A-8 reached 120 g.l-1.Then,haploid strain SM-4 from M-3 with high biomass and SA-5 from A-8 with high ethanol concentration were slected as parental strains for protoplast fusion.The optimum conditions of formation and regeneration of both parental strains were as below:the cells were pretreated with a mixture of 25ml 0.05mol/L EDTA and 1ml 0.5mol/lβ-ME for 1h,then cell walls were broke with 4%snailase for 1.5h.The protoplast formation rate and regeneration rate of SM-4 were 89.3%and 22.6%respectively,and for SA-5,they were 91.45%and 17.9%.The protoplast of SM-4 was inactivated with UV,the optimum conditions were:treating for 4min below the UV lamp(20W).The protoplast of SA-5 was inactivated with heating,the optimum conditions were:treating for 30min in electric-heated thermostatic water bath(65℃).The inactivated protoplast fusion was induced by PEG.We obtained a fusant F-13 which can produce ethanol at a high concentration after TTC screening,fermentation and 5-batches inheritance stability study.The biomass reached 26g.1-1 with its ethanol concentration in fermentation broth 135 g.1-1,which 7.3%and 17%higher than SA-5 and SM-4 respectively,the ethanol tolerance capacity achieved 160 g.1-1.Utilizing methods above,protoplast fusion was accomplished between haploid strain 1770 S-5 of Pachysolen tannophilus and SM-4 after heat and UV inactivation,respectively.A fusant F-9 was abtained after TTC screening,fermentation and 5-batches inheritance stability study.Its ethanol concentration in fermentation broth was 25.55 g.1-1 with ethanol yield 0.25 g.g-1.
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