Microbial Tolerance Mechanism And Application Involved In The Inhibitors Of Biomass Pretreatment

Using lignocellulosic biomass for the production of renewable biofuel and value-added chemicals provides a sustainable and promising solution to the crisis of energy and environment.However,pretreatment process would bring inhibitors in biomass slurry.The inhibitors left in hydrolysate would inhibit cell growth and fermentation process.Therefore,it is crucial to reveal the tolerant mechanism and to develop tolerant strains for biofuel production.In this research,we studid the tolerance,metabolic responses and tolerant mechanism of Candida tropicalis and Escherichia coli in response to pretreatment inhibitors.The main results are as follows:(1)We analyzed the inhibition mechanisms and the metabolic responses to complex inhibitors(CI,including furfural,phenol and acetic acid)in hydrolysate.We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment indicating that the flux from xylulose to pentose phosphate pathway increased and less xylitol was accumulated.Moreover,the changes in levels of 1,3-propanediol,trehalose,saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress.The increase of 1,3-propanediol level was considered to be correlated with regulating redox balance and osmoregulation.The increase of trehalose level might play a role in protein stabilization and cellular membranes protection.Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane.The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource.(2)In this study,C.tropicalis showed a strong ability of furfural tolerance and furfural degradation during fermentation.C.tropicalis’ half maximal inhibitory concentration for furfural with xylose as the sole carbon source at 24 h was 3.69g/L,which was higher than that of most wild-type microbes reported in literatures to our knowledge.when adding furfural of 3,5 and 7g/L into the culture at mid-exponential phase(6h after inocullation),the rate of furfural degradation reached 3,3.33,and 2.80gL-1h-1.Mechanisms of furfural detoxification and metabolic responses in the main pathways were revealed by multiple analyses.Alcohol dehydrogenase 1 was confirmed to be responsible for furfural detoxification.By real-time PCR analysis,we found that the expression of ADH1 in C.tropicalis(ctADHl)from was induced by furfural and repressed by ethanol after furfural depletion to maintain redox balance.The expression of ctADH1 could be regulated by both furfural and ethanol.C.tropicalis showed a complex regulation system during furfural detoxification to minimize adverse effects and oxidative stress caused by furfural.The study provides valuable insights into tolerance enhancement and strain modification for efficient lignocellulose fermentation.The rate of furfural degradation in E.coli BL21(DE3)with pET-ADH1(high-copy plasmid)and pCS-ADH1(medium-copy plasmid)was increased 1.59-fold and 1.28-fold,respectively.The mechanism we uncovered in this work was successfully applied to enhance E.coli’s furfural tolerance by heterologous expression of ctADH1.The application also further proved the function of ctADH1.(3)In this study,we evaluated the effect of carbon sources(xylose,glucose and glucose-xylose)on furfural tolerance,furfural degradation and redox balance.We found that the rate of furfural degradation and half maximal inhibitory concentration for furfural of C.tropicalis in xylose medium increased 1.68-fold and 1.19-fold,respectively,compared with those in glucose medium,indicating that C.tropicalis obtained better furfural tolerance in xylose medium.The dehydrogenation of xylitol,which produces coenzyme NADH,promotes the recycle of NAD+ and facilitates the reduction of furfural.This study provides important information for metabolic regulation and metabolic engineering of efficient lignocellulose fermentation strains.(4)In this study,we characterized the growth and biofuel production of IL-tolerant E.coli obtained by adaptive laboratory evolution.Adapted E.coli showed increased biomass,increased IC50 and increased biofuel production compared to the parent strain.The growth of E.coli MG1655-A1 was even promoted by[BMIM]Cl and[EMIM]Cl of specific concentrations.E.coli MG1655-A1’s isoprenol titer in the presence of 100mM[BMIM]Cl,100mM[BMIM]Ac,300mM[EMIM]Cl and l00mM[EMIM]Ac reached 0.60g/L,0.22g/L,0.49g/L and 0.56g/L,respectively.We also found that E.coli MG1655-A1 was a promising host to use the acetate of[BMIM]Ac in wastewater for growth biofuel production.E.coli A1’s isoprenol titer using 50mM[BMIM]Ac as carbon source reached the level of that using 50mM NaAc.The adapted E.coli provided new host strains for one-pot biofuel production integrating IL-based pretreatment with saccharification and fermentation and also provided an approach to reuse and recycle ILs in wastewater.(5)In this study,NaCl was found to enhance E.coli’s tolerance to[BMIM]Cl,[BMIM]Ac and[EMIM]Cl without modifying the host bacteria.NaCl could also improve E.coli’s biofuel production in the presence of[BMIM]Cl,[BMIM]Ac,[EMIM]C1 and[EMIM]Ac.E.coli DH1’s isoprenol production in the presence of 50mM[BMIM]Cl,50mM[BMIM]Ac,50mM[EMIM]Cl and 50mM[EMIM]Ac was increased 2.0-fold,3.3-fold,13.1-fold and 1.5-fold by 200mM NaCl,respectively.NaCl was found to significantly improve E.coli’s growth and biofuel production in the presence of ILs,providing a new simple strategy to enhance E.coli’s tolerance to ILs and fermentation performance.This strategy was cost-effective and did not need modifying strains,provides a new approach to integrate biofuel fermentation with IL-pretreatment and saccharification as one-pot process.