Screening Of Thermotolerant Yeast Strains And Ethanol Fermentation Of Molasses

Ethanol as a green and renewable energy is becoming a new energy in various countries in the context of deteriorating environment and energy crisis. Microorganism fermentation is the main ethanol production approach. In industrial ethanol fermentation, the fermentation temperature is30-33癈, but, due to seasonal and environmental factors and yeast’s heat production, temperature at the later stage of fermentation can reach38癈. To ensure normal growth of yeast, cooling measures must be taken, which will consume large quantity of energy and increase the cost of ethanol production. For this reason, the screening of thermotolerant yeast strains for industrial ethanol production is of great significance.In this study, a total of20yeast strains were selected from the soil samples of Xishuangbanna Tropical Botanical Garden. S16yeast strain, which was tolerant to48癈and capable of high-yield ethanol fermentation at48癈, was screened.Morphological, physiological and biochemical assays of S16yeast strain showed: S16yeast strain was asexually reproduced by single budding with germination of oval ascospore. Carbon sources that could be assimilated by S16yeast strain included raffinose, glucose, sucrose, maltose, galactose, lactose, D-xylose, D-mannitol, D-sorbitol, DL-lactic acid, glycerol, ethanol and citrate; S16yeast strain could not assimilate L-rhamnose, L-sorbose, soluble starch, xylitol, cellobiose and erythritol; S16yeast strain was unable to assimilate nitrogen source such as nitrate but able to assimilate ethylamine; it could grow on vitamin-free medium or on YPD medium supplemented with60%glucose; it had no urease activity; and it was able to grow in0.1%and0.01%Chlorhexidine Dihydrochloride. Referring to Yeast Characteristics and Identification by JA Barnett published in1991, we identified S16yeast strain as Kluyveromyces marxianus of genus Kluyveromyces.S16yeast strain was subjected to molecular identification in D1/D2+ITS region. Result indicated that the sequence of this region after amplification had99%similarity with that of Kluyveromyces marxianusCHY1612in GenkBank. Thus, we confirmed that S16yeast strain was Kluyveromyces marxianus of genus Kluyveromyces on molecular level. Basic physiological research of S16yeast strain indicated that the appropriate temperature for the growth of S16yeast strain was25癈-42癈. On both liquid and solid media, the highest temperature that S16yeast strain was tolerant to was48癈, with appropriate pH value ranging from3to7. At fermentation temperature of42癈, the S16yeast strain’s ethanol tolerance was6%, and its salt tolerance was6%.We preliminarily researched the characteristics of ethanol fermentation by S16yeast strain from glucose and sucrose, and optimized the fermentation conditions. The results showed that, when glucose was fermentation substrate, the highest ethanol concentration within the range30癈-48癈was unaffected by temperature. However, the ethanol production rate varied significantly, which reached2.85g/1/h at30癈and3.792g/l/h at42癈. At48癈, the ethanol concentration rate fell back to1.826g/1/h. At10h, the conversion rate of glucose into ethanol reached the highest of80.96%at42癈, and the volume fraction of ethanol in fermentation broth reached4.8%. But, when sucrose was fermentation substrate, the ethanol production rate reached the highest value of3.389g/1/h at12h, and the conversion rate of sucrose into ethanol was79.6%. The highest conversion rate of sucrose into ethanol was91.3%, and the volume fraction of ethanol in fermentation broth reached6.2%. Therefore, the optimal fermentation temperature for S16yeast strain on both glucose and sucrose was determined as42癈.Next, we researched ethanol fermentation by S16yeast strain from cane molasses and optimized the fermentation conditions. By single-factor experiment, the optimal brix for S16yeast strain in ethanol fermentation from molasses was20BXo, with the addition of magnesium sulfate and yeast extract being0.02%and0.5%, respectively. After orthogonal experimental design, we set the optimal initial brix to be20BX for S16yeast strain in ethanol fermentation from cane molasses, in order to obtain the highest ethanol yield; the addition of yeast extract and magnesium sulfate was respectively0.5%and0.04%. Then, shake-flask fermentation was carried out for ethanol fermentation from molasses under optimized conditions. Compared with Angel yeast, a thermotolerant yeast strain in industrial ethanol fermentation, the ethanol production rate of S16yeast strain within12h was significantly higher than that of Angel yeast; at12h, the ethanol production rate of S16yeast strain was3.3g/1/h, and that of Angel yeast,2.66g/1/h. The conversion rate of molasses to ethanol for S16yeast strain, which reached92.27%of the theoretical value, was also higher than that for Angel yeast.Waste fermentation broth from molasses may cause environmental pollution if not properly treated. In this study, we also studied the recycling technology for waste fermentation broth. Waste fermentation broth could be re-used in the dilution of molasses to replace clear water. Waste fermentation broth from molasses was added into molasses liquid at different proportions, and we found that such addition influenced the growth of S16yeast strain to variable degrees at different proportions of waste fermentation broth. When the proportion was below75%, the yield of S16yeast strain and ethanol yield was very little influenced. Therefore, waste fermentation broth could be re-used to dilate molasses liquid at the addition proportion below75%, in order to meet the requirements of clean ethanol production.