Optimization Of Fermentation Conditions And Breeding Of Cellulosic Ethanol Production Strain Spathaspora Passalidarum

Fuel ethanol is considered to be able to replace traditional fossil energy and is the most mature and widely used bioenergy.Different from the first generation of fuel ethanol,the second generation of lignocellulosic ethanol has more obvious advantages.However,there are still a series of limitations in the production of cellulosic ethanol,such as low conversion of sugar and alcohol,long fermentation time,inhibitors affecting fermentation and so on.The comprehensive utilization of mixed sugars in lignocellulose hydrolysate and the improvement of tolerance to fermentation inhibitors in lignocellulose hydrolysate have always been the key issues in the utilization of lignocellulose hydrolysate.Spathaspora passalidarum,as an unconventional yeast,has the ability of natural co-fermentation of xylose and glucose.In this study,two aeration strategies were used to further improve the fermentation ability of the strain.50g/L mixed sugar(glucose mass: xylose mass = 1:1),when no hydrolysis inhibitor was added,the fermentation ended at 36 h,and the sugar-alcohol conversion rate was 0.46 g/g.After adding lignocellulose hydrolysis inhibitor,the fermentation could not be finished for 96 h,and the sugar-alcohol conversion was only 0.09 g/g,and xylose could hardly be utilized.Using ORP(Oxidation-Reduction Potential)control strategy,when the optimal ORP setting value is-110 m V,the fermentation finished in 48 hours,and the conversion rate of sugar to alcohol is0.47 g.Constant ventilation could also promote the fermentation process of mixed sugar with hydrolytic inhibitors,but the effect was worse than that of ORP control strategy.Then the effect of pH on fermentation performance was investigated at shaking flask level.S.passalidarum could consume 50 g/L xylose in 72 hours under initial conditions,and the yield of ethanol was 0.32 g/g.After adding mixed inhibitor,50 g/L xylose was still not exhausted for96 hours.Therefore,adjusting the fermentation medium pH from 7 to 3 before sterilization.When pH decreased to 3,the xylose depletion time was shortened to 24 hours,and the conversion rate of alcohol to sugar was increased to 0.46 g/g.Then sterilization was carried out after adjusting the fermentation medium pH to 3 with different acids.It was found that strong acids could improve the fermentation ability of S.passalidarum,but acetate had no promoting effect on fermentation.The concentration of toxic substances produced by Maillard reaction could be greatly reduced and the fermentation performance could be improved by regulating pH with strong acids.Finally,the effects of different carbon source ratio and the addition of redox agent on the fermentation ability of S.passalidarum were investigated.It was found that the presence of a small amount of glucose could promote the metabolism of xylose by S.passalidarum and achieved co-fermentation of glucose/xylose.The addition of anhydrous sodium sulfite could increase the conversion rate of sugar and with 0.37 g/g productivity.After adding the inhibitor,the adverse effect of sodium sulfite on the inhibitor was alleviated,the fermentation performance was improved,and the conversion rate of sugar and alcohol was increased to 0.39g/g at 1.5 g/L.On the other hand,in order to obtain the strain with strong tolerance,a strain with improved ethanol tolerance and acetic acid tolerance was obtained by ARTP mutagenesis and long-term domestication,and the ethanol tolerance was increased from 32 g/L to 72 g/L,the acetic acid tolerance was improved from 2.6 g/L to 4.4 g/L.