High-Solid Enzymatic Hydrolysis Of Sugarcane Bagasse And Co-Fermentation Of Mixed Sugars In Bioethanol

In recent years,the technical route of cellulosic ethanol production has been successfully constructed,but it still lacks of economic feasibility as it cannot approach the “three high” levels(high titer,high conversion rate,high productivity)of the current starchy ethanol fermentation industry.In this thesis,the high-solid enzymatic hydrolysis of atmospheric glycerol organosolv pretreated sugarcane bagasse with a low cellulase loading and ethanol co-fermentation on mixed sugars released from the hydrolysate using engineering yeast were carried out.The detail of the contents is as follows:First,the fermentation performance of the engineered Saccharomyces cerevisiae was evaluated.The optimal fermentation conditions were determined: temperature 30 °C,inoculum10%(v/v),rotational speed 180 rpm,and initial p H 5.5.Under these conditions,the ethanol titer of 85.1% was achieved.Then,single sugar fermentations,as well as mixed sugar fermentation with different ratios,were evaluated and the results showed that the strain could efficiently utilize xylose to ethanol,and a better-mixed sugar ratio(glucose / xylose = 2/1)was determined.The above assessment laid a solid foundation for the subsequent glucose-xylose co-fermentation by this strain to produce ethanol.Second,high-solids enzymatic hydrolysis process of alkali-catalyzed atmospheric glycerol organosolv(al-AGO)pretreated sugarcane bagasse was explored.With the principle of “high solid content,low enzyme loading”,the fed-batch mode was used to run the high-solid enzymatic hydrolysis.The fed-batch mode for the enzymatic hydrolysis at 20% solid content with 6 FPU/g of cellulase loading was fixed as follows: 7% of the initial substrate and 5%,3%and 5% of feeding at 6 h,12 h and 24 h,respectively.This enzymatic hydrolysis released about150 g/L of fermentable sugars,with 69.6% of the glucose yield after 72 h.Further,the solid content increased to 35% for the enzymatic hydrolysis at 3 FPU/g of enzyme loading,which was selected as follows: 16% of the initial substrate,and 7%,6% and 6% of substrate feeding at 12 h,24 h and 36 h,respectively.The enzymatic hydrolysis reached 200 g/L of the fermentable sugars containing 141.1 g/L of the glucose titer and 69.5 g/L of the xylose titer for72 h.Finally,the ethanol co-fermentation on these fermentable sugars in enzymatic hydrolysate was explored.The different fermentation processes and batch / fed-batch co-fermentation methods in shake flasks was studied.Results showed that the separate hydrolysis and cofermentation(SHCF)process was desired.With the fed-batch mode,the ethanol fermentation was carried out initially with 150 g/L of fermentable sugars and fed with 50 g/L mixed sugars at 12 h.The fermentation produced 73.8 g/L of the ethanol titer for 72 h and correspondingly74.1% of the ethanol yield.The experiment was tested in a 5-L fermenter,in which the fermentation on the synthetic medium started with 150 g/L(glucose / xylose = 2/1)of initial mixed sugars and was with 50 g/L of mixed sugars twice at 10 h and 21 h,respectively.The fermentation achieved 99.6 g/L of the ethanol titer and 89.7% of the yield for 72 h.With the enzymatic hydrolysate for alternative to the synthetic medium,cellulosic ethanol fermentation under the same condition produced 86.5 g/L of the ethanol titer and 84.1% of the yield for 72 h,respectively.The fed-batch mode was changed based on the xylose consumption rate,so the ratio of glucose and xylose was adjusted to be 2.66/1(250 g/L of the total sugar titer).After 72 h of the fermentation time,the ethanol titer reached 102.2 g/L with 86.6% of the corresponding ethanol yield.In this thesis,significant progress was made at the high-solid enzymatic saccharification.The “three-high” level of cellulosic ethanol production was very close to that of the ongoing starch sugar ethanol fermentation,which provides a technical reference for the industrialization of cellulosic ethanol.