Isolation, Identification Of Thermotolerant And Building Thermotolerant High-producing Yeast By Protoplast Fusion

In different environments, 227 isolates out of were screened for fuel ethanol-producing yeast with high thermotolerance. Among them, the strain h-1, which was able to use maize as fermentation substate to produce higher alcohol in 42 ℃, was identified as Hansenula Saturnus based on its biophysiological and biochemical characteristics, and employed for the following fusion.Chemical drugs and UV which exterminate singal parent's protoplasts were choosen as hereditary markers. 0.01% tradimefon could inhibit the strain SP-48, but couldn't inhibit h-1. So, tradimefon whose content was 0.01% was choosen as marker. The relation of UV's irradiated dosage and h-l's protoplasts lethal ration was identified. When UV light was 20W, the irradiated distant was 30cm, and the time was 2min, the lethal ration of h-l's protoplasts was 100%. So, tradimefon whose content was 0.01% and UV whose irradiated h-1 2min were used as hereditary markers. The protoplast yeasts of h-1 and SP-48 were fused using PEG, and fusion strains LZ-3 which could tolerate high-temperture and produced higher alcohol was achieved. After 72h fermentation, the fusion strains LZ-3 could produce alcohol's ration 7.5%. The fusion strain had stable heredity, and when inoculated 20 times, alcohol-producing ration of the fusion strain LZ-3 was not obvious changes.The fusion yeasts LZ-3 grew the best in 36℃ and a pH of 5.0,whereas its optimum fermentation temperature and pH was 34℃ and 5.0,respectively.It was seen that temperature and initial inoculum's concentration were 2 important factors in affecting the growth and fermentation of strain 3.The strains of 3 showed good tolerances to various stresses including aciding, alkalis, and osmotic pressure. Also, the strains of LZ-3 showed .better resistances to high concentration alcohol and byproducts than the produced-commercially ADY(acid dried yeast).If 33.3%(w/v)concentration of crude corn in fermenting material, 400 units alucoamylse per gram of raw corn, 9%(v/v) inoculum size of the yeast, and initial pH of 5.5 were fixed, orthogonal test indicated that the fusion strain LZ-3 produced the highest amount of alcohol and 9.0% after incubated at 42℃ for 72 hours when it was tested in higher temperature.