Strains Construction For Cellulosic Ethanol Production And The Optimization Of Fermentation

Fuel ethanol is currently the fastest growing bioenergy source.Cellulosic raw materials are widely available and inexpensive.Meanwhile,efficient conversion of cellulosic raw materials is also beneficial to the rural economy.Therefore,cellulosic ethanol has received worldwide attention.However,there are some technical problems in the production process of cellulosic ethanol: no strains fermenting glucose and xylose efficiently,multiple hydrolyzate inhibitors present and they will influence the fermentation efficiency.In this study,Kluyveromyces marxianus,potential in cellulosic ethanol production was selected to study the new fermentation strategy of cellulosic ethanol fermentation,and the original xylose metabolic pathway was modified by genetic engineering to improve ethanol production and shorten the fermentation time.Firstly,the glucose and pentose fermentation capacity,high temperature tolerance and inhibitor tolerance were compared by the new candidate in cellulosic ethanol production,Spathaspora passalidarum MYA-4345,and the Kluyveromyces marxianus 1727-5,domesticated by our laboratory.Although S.passalidarum MYA-4345 showed better xylose fermentation performance and higher ethanol yield,the fermentation performance under higher temperature was strongly inhibited.K.marxianus 1727-5 showed better fermentation capacity of glucose and arabinose,which also remained high efficiency at high temperature.Meanwhile,K.marxianus 1727-5 was more resistant to acetic acid and formic acid,even had the ability to grow in the concentration of 15 g/L acetic acid.Moreover,K.marxianus 1727-5 also showed stronger fermentation ability in the simultaneous saccharification cofermentation using the undetoxified corncob hydrolysates.According to the results above,K.marxianus 1727-5 was selected as the host cell in the follow-up experiments.Based on the fermentation characteristics of K.marxianus 1727-5 and the problems existing in the cellulosic ethanol fermentation process,this study developed a new fermentation strategy: two-step fermentation strategy.In the first step,xylose of the cellulosic material was directly fermented after diluted-acid pretreatment without detoxification,the micro-aeration strategy was used to alleviate the inhibition of glucose;and after cellulose adding,the second step was the ethanol production from glucose in the micro-aeration condition.The aeration rate of the fermentation process in the first step and the cellulase dosage in the second step were optimized.The optimal fermentation condition was: aeration volume of 1.4 vvm,cellulase dosage of 10 FPU/g,and the cellulase addition time of 60 h.Under the optimal condition,the xylitol yield was 0.7 g/g,the ethanol yield was 0.41 g/g,and the fermentation time was 96 h.The results indicateed that K.marxianus 1727-5 and two-step fermentation strategy were potential in the industrial process of cellulosic ethanol production.In order to further improve the yield of xylose to ethanol,the xylose metabolic pathway was genetically engineered in K.marxianus 1727-5.It was found that the fermentation rate of xylose and ethanol yield were both improved by the recombinant strain expressing XYL1.2,XDH and XK together,which proved that XYL1.2,XDH and XK played important roles in the metabolism of xylose.Using the optimized two-step fermentation strategy,the xylitol yield was 0.8 g/g,the ethanol yield was 0.44 g/g,and the fermentation time was 90 h by the recombinant strain.In summary,this study lay the foundation for cellulosic ethanol production in the strains and fermentation processes.