Effects Of Inhibitors From Lignocellulose Pretreatment On Ethanol Fermentation Of The Flocculating Yeast SPSC01

Fuel ethanol is a liquid fuel that widely used and studied in the world,which could alleviate the energy crisis and environmental pollution.Lignocellulosic biomass is the cheap and abundant source of raw materials for large-scale production of bioethanol,meanwhile,Saccharomyces cerevisiae has been applied on winemaking,baking,and brewing since ancient times.The toxic by-products produced from biomass pretreatment processes,such as furfural and 5-hydroxymethyl-furfural(5-HMF),could severely inhibit the yeast growth and the ethanol fermentation.Therefore,in fact,constructing or improving the stress tolerant yeast strains by revealing the synergism among toxic inhibitors is of great significance.In this work,the flocculating S.cerevisiae SPSC01,and its FLO1 gene deleting mutator(3141)as a reference strain were employed for comparing the stress tolerance in the representative inhibitors derived from the classification of furan compounds,phenols,and organic acids.This research focused on effects of individual inhibitors and synergistic inhibition on cell growth and ethanol production of industrial yeast strains with flocculating and non-flocculating phenotype.Except 5-HMF,the,SPSC01 could tolerant high concentrations of formic acid,acetic acid,furfural vanillic acid and vanillin when compared with 3141 mutator,and the inhibitory tendency of cell growth was coincident with the trend of ethanol production in the SPSC01 and 3141 strains.Further analysis of the byproducts in fermentation broth showed that concentration of succinic acid and glycerol decreased when the inhibitors were supplemented,and the reduction degree of byproducts of SPSC01 were lower than that of 3141,which inhibition trend was similar to the yeasts growth.Secondly,the synergistic effect of formic acid and 5-HMF(SPSC01:5.48,3141:6.85,the mole ratio),vanillic acid and furfural(SPSC01:1.14,3141:1.14,the mole ratio)on yeast growth was obtained respectivily.And the tolerance to the inhibitors of SPSC01 was higher than 3141 when the inhibition rate was more than 50% under the synergy.metabolic byproducts in the fermentation broth were analyzed and showed that the concentration of glycerol and succinic acid were lower than that of the individual inhibitors under the synergy.Furthermore,the activities of SOD,CAT,ADH and ATP concentration in the yeasts cell were measured,the activity of all enzymes in the yeasts cells were lower than that of the control.And 5-HMF and formic acid had the synergistic inhibitory effect on the ATP and ADH activity of SPSC01 and 3141,however,the stronger synergistic inhibition was obtained on 3141.Finally,overexpression of sphingosine long chain base kinase gene LCB4(YOR171C)can improve the tolerance of model yeast S288 c to furfural,vanillin and acetic acid,which can also shorten the fermentation time and improved ethanol yield.Compared with the individual inhibitors,the synergistic inhibitory effect of furfural,vanillin and acetic acid on the yeasts cells growth was significant.The above studies mirrored the relationship between stress tolerance and flocculating yeast in biomass accumulation,ethanol production,metabolites,which provides guiding basis for strain genetic breeding and real lignocellulosic ethanol production.