Improving Ethanol Yield Of Saccharomyces Cerevisiae Strains For Very High Gravity Fermentation By Reduction Of Glycerol Formation And Expression Of ?-glucosidase

Now fuel ethanol is widely used all over the world and is one of the most important bio-fuel.It is necessary to make full use of all kinds of biomass,and to increase the bio-ethanol yield and decrease the fermentation cost.Very high gravity fermentation,simplified as VHG technology,is proved to efficiently decrease the energy consumption as well as producing cost.But,at the same time,it would lead to a challenge on fermentation strain and process.It is a key problem in genetic modification of fermentation strain,to how to realize a rapid growth and fermentation and increase raw material utilization ratio for highly efficient fermentation.For this target,those researches have been carried out as followings in this study.VHG fermentation evaluation of corn material by genetically modified yeast strains from previous work in our lab,which were proved to reduce glycerol formation and improve the ethanol yield.The results of anaerobic fermentation test with 200?450 g/L glucose in YPD showed that the stain Ka-4#received the highest ethanol yield 97.04%with the 300 g/L glucose;based on this,30%solid content of corn slurry,liquefaction and saccharification processing procedure were determined.Orthogonal design of experiments were further designed with three factors and four levels,and the results indicated that the optical combination was strain Ka-5#,1 g/L urea and 4 g/L yeast extracts.Further flask evaluating showed that the maximum ethanol concentrations during 72 h fermentation time were 136.3?142.9 and 132.7 g/L for strains Ka-4#,Ka-5#and TH-AADY,respectively.The corresponding maximum glycerol concentrations were 7.8,7.9 and 10.9 g/L,respectively.On the other hand,the strain Ka-4#was proved to ferment more quickly and the ethanol titer at 24 h was up to 95.3%of the maximum value during the whole course.The mating between strain K?-4#from strain Ka-4#and Ka-5#was carried out,the resultant diploid strains,named by Ka?-1#?Ka?-10#,were screened and tested by corn VHG fermentation evaluation.The fermentation results demonstrated that the strain Ka?-5#was the best among them,which combined the advantages of the parental strains.The?-glucosidase-expressing strains were constructed by delta-integrating method and their enzymatic activities were determined.The delta-integrating plasmid p G?RU-BGL was first constructed with delta sequences as homologous arms,then linearized by enzymatic digestion and transformed into strain K?,which was derived from industrial yeast.The result?-glucosidase-expressing strains were named by K?-BG1?K?-BG7.The activity determination indicated that the strains K?-BG2 and K?-BG6 had higher total enzymatic values than others,0.24 and 0.27 U/m L/OD₆₀₀,respectively.Three kinds of the strains aforementioned were all evaluated under the same conditions by using corn and cassava materials.1)The process procedure special for cassava was first designed and selected.Then the effect of solid content on final ethanol titer and yield was investigated by using strain Ka-4#.The results showed that35%solid content led to the highest ethanol concentration value 165.7 g/L,but 32.5%solid content resulted to the highest ethanol yield 89.3%.2)The effect of?-glucosidase addition was investigated during the saccharification step of 30%solid content corn or 35%solid content cassava.The results showed that for the cassava slurry,three dealt methods with adding glucoamylase,glucoamylase puls?-glucosidase and?-glucosidase,respectively,would lead to 375,415 and 210 g/L glucose,respectively.For the corn slurry,the values were 230,252 and 148 g/L,respectively.Those data implied the helpful and effective function of?-glucosidase on glucose release from corn or cassava.3)The fermentation performance was compared among the three strains with 32.5%solid content cassava and 30%solid content corn,and the results indicated that the ethanol titer of strain K?-BG2 was 3.34%or 2.33%higher than that of strain K?-BG6.Therefore the four strains,Ka-4#,Ka?-5#,K?-BG2 and TH-AADY,were selected to together be used in 32.5%solid content cassava and 30%solid content corn fermentation.The results showed that for the cassava slurry,the maximum ethanol concentrations during 72 h fermentation time were 159.6,167.0,163.5 and 155.8 g/L,respectively.The corresponding maximum glycerol concentrations were 9.23,10.17,12.06 and 12.96 g/L,respectively.Again,the maximum ethanol yield values were 87.8,91.6,88.7 and 84.8%,respectively.The strains except K?-BG2 obtained the 1.89%?3.86%increasing of ethanol titer by additionally supplementing?-glucosidase into slurry systems.In summary,those data well proved the genetically modified yeast with reducing glycerol formation could apply for VHG fermentation process of corn or cassava.The strategies to combine the advantages between strains with different and excellent performance characteristics and to express?-glucosidase,could make the VHG fermentation performance better by leading to higher ethanol-producing rate,or/and higher ethanol titer and yield.