Strategies For Ethanol Fermentation From Jerusalem Artichoke And Regulatory Mechanisms Of Inulinase Expression

Bioenergy contributes to alleviating the energy crisis and promoting the sustainable development of human beings.Industrial-scale production of fuel ethanol may lay a powerful foundation for the model transformation of energy consumption.Ethanol production from non-grain feedstock represents a great promise for countries like China.Currently,Jerusalem artichoke(JA)attracts increasing attentions due to its advantages in high yield,resistance to poor soil,drought,cold temperature and pests.Compared with traditional starch-based materials,ethanol fermentation from JA is blocked by long fermentative time and high residual sugar concentrations.Therefore,in this study,engineered strategies and the detailed regulatory mechanisms of inulinase expression were investigated to optimize the process of ethanol fermentation from JA by Kluyveromyces marxianus,which may provide theoretical reference for its futher industrialization.Firstly,effects of continuous aeration on ethanol fermentation from JA by K.marxianus Y179 were evaluated.Continuous aeration(0.25 and 0.5 vvm)significantly accelerated this process with low concentrations of residual sugar,but concentrations and yields of ethanol decreased.Relatively anaerobic conditions were more suitable for ethanol fermentation,but long fermentative duration,limited inulinase activities and high concentrations of residual sugar were also observed;To solve the contradiction between aeration and high ethanol yield,"two-stage aeration strategy",keeping the aeration at 0.25 vvm at the first 12 h,and droping it to 0.06 vvm,was subsequently proposed,which shortened fermentative time by over 36 h,and decreased residual sugar concentration to less than 12 g/L,achieving ethanol concentration and productivity of 97.97 g/L and 1.63 g/L/h,respectively.On this basis,fed-batch ethanol fermentation from raw JA tubers was conducted under the control of "two-stage aeration strategy",which eventually showed a greatly accelerated fermentative process,with the relatively high concentration and yield of ethanol,and low concentration of residual sugar(less than 10 g/L),compared with anaerobic conditions.Secondly,effects of oxidation-reduction potential(ORP)control on ethanol production were investigated.ORP control is supposed to be a more efficient and effective aeration strategy for ethanol fermentation from JA by K.marxianus Y179.Compared with precious "two-stage aeration strategy",higher productivity(1.63 and 2.04 g/L/h)and lower concentration of residual sugar(12 and 6 g/L)were achieved when ORP was controlled at-130 mV;At the same time,the positive impact of aeration on ethanol production might be attributed to the enhanced inulinase activities and cell viabilities,by examining effects of extra addition of inulinase and enhanced initial biomass on fermentative performances.Besides,transcriptional analysis was conducted during ethanol fermentation by K.marxianus to analyze the differences in gene expression between high and low sugar concentrations,and between aerobic and anaerobic conditions,respectively.Differentially expressed genes have been proved to involve in central carbon metabolism,hexose transportors and oxidative stress defense,ect.In particular,genes related to oxidative stress defense and autophagy promoted cell survival under harshest conditions.Furthermore,effect of some selected genes on enhanced fermentative profiles and cell survival were validated.Oxidordeuctases that were represented by KmTPXl,KmTrxR and KmTRX greatly promotes the cell growth and ethanol formation under hard conditions,which explained the reason for an promoted fermentative profiles by K.marxianus under conditions of high sugar concentration and high oxidative stress;More importantly,these oxidordeuctases,especially KmTPX1,enhanced tolerance of yeasts to multiple stressors like H2O2,formate,acetate and ethanol,and promoted ethanol fermentative performaces with inhibitors,which further established basis for applications in the field of lignocellulosic ethanol.Finally,a detailed regulatory mechanisms of inulinase expression from K.marxianus were explored.On the one hand,inulinase activities was induced by inulin,and repressed by glucose and high concentrations of carbon sources.The aeration and ethanol mainly affected cell growth but not inulinase activities.On the other hand,inulinase promoter with the total length of 1053 bp was targeted to expound its regulation by carbon sources.Results showed that the promoters with different lengths were all induced by inulin and inhibited by high glucose concentration,in spite of the differences in activities.The inulinase promoter with a length of 353 bp showed higheast activity with weakest responses to high glucose concentration,and further experiments identified the minimal functional region extending from-271 bp to-110 bp.And the responses of inulinase promoter to carbon source have been proved to be related to the Migl binding-site extending from-496 to-485 bp.Mutated promoters with double activities by random mutagenesis broaden its possible applications in intensive inulinase activities.Above all,strategies like "two-stage aeration" and "ORP control" contribute to the process of ethanol fermentation from JA,achieving the accelerated fermentative duration and low residual sugar concentrations.Transcriptional analysis revealed the theoretical basis of aeration promoting fermentative process.A comprehensive understanding of regulatory mechanisms of inulinase expression by carbon sources at transcriptional levels contributes to further studies on its strone activities and responses to carbon sources.Eventually,construction of a recombinant K.marxianus strain with the well-controlled inulinase expression may promote the process of industrialization for ethanol production from JA in the future.