Derived Feature Of Phenolics During Acid Pretreatment Of Corn Stover And Its Inhibitory Mechanism To Butanol Fermentation

Biobutanol is a bio-fuel with high energy value and low corrosion.Using lignocellulosic biomass as raw material to produce butanol has the advantages of low raw material cost and friendly environmental performance.Pretreatment is the key procedure to achieve efficient biotransformation of lignocellulosic,but many inhibitors derived during this process are one of the bottlenecks limiting the efficient synthesis of butanol.Phenolics derived from lignin have strong inhibitory effects on solventogenic clostridia.In addition,it is hard to measure the kinds and concentration in the biomass hydrolysate of phenolics with their poor water solubility and various phenolic compounds.Meanwhile,the derived feature of phenolics during the pretreatment and the inhibitory mechanism to butanol fermentation are unclear,which make the production of butanol from lignocellulose lacking rational guidance.Based on the above problems,in this thesis,corn stover was selected as lignocellulose to analyze the derived feature of phenolics during acid pretreatment.The global effect of the co-existence of phenolic acids and phenolic aldehydes on butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated,and the molecular mechanism of such substances inhibiting butanol fermentation was elucidated.The major conclusions were summarized as follows:(1)The interaction between different phenolic compounds and cellulose enzymatic hydrolysis were studied,and the influence of different acid pretreatment processes on the derived phenolic contents(CPhe)was investigated.The results showed that with the increase of pretreatment temperature,the glucose yield(CGlc)gradually increased.When the pretreatment temperature reached to 180～200℃,the cellulose components degraded excessively,which resulting in a lower CGlc.In addition,the CPhe in the hydrolysate changed,and 1 g/L phenolics can reduce the enzymatic hydrolysis effect significantly.(2)An artificial neural network model was established to predict the derived features of CPhe and CGlc in the hydrolysate pretreated by dilute inorganic acid.The results showed that the artificial neural network model had a good fitting performance(R2>0.90)for the prediction of CPhe and CGlc.The relative importance of inorganic acid kinds(kIA),concentration of inorganic acid(CIA),pretreatment temperature(T),pretreatment time(t),solid-to-liquid ratio(RSL),and enzyme loading dasage(E)on CPhe and CGlc was calculated.The results showed that the relative importance of five parameters(CIA,T,t,RSL,and kIA)on CGlc accounts for 23%,14%,12%,16%,and 14%.Enzyme dasage(E)accounts for 21%importance on CGlc.In addition,CIA had the strongest importance on CGlc and CPhe,which reaching 23%and 22%,respectively.(3)Influences of combined phenolic acids/aldehydes(Com-Phe)to butanol fermentation of C.acetobutylicum ATCC 824 were studied.The apparent kinetics of butanol fermentation performance showed that the glucose utilization rate decreased by 21.2%after Com-Phe stress for 12 h in comparison with the control,and finally produced with a low butanol titer of 6.4 g/L.Furthermore,compared with the control,the intracellular reactive oxygen species(ROS)levels of C.acetobutylicum under phenolic acids,phenolic aldehydes,and Com-Phe stress were enhanced by 1.04-,2.40-,and 1.68-fold,respectively.The results showed that inhibitory effect of Com-Phe on solvents biosynthesis was stronger than that of phenolic acids,but weaker than phenolic aldehydes.(4)The transcriptome profiles of the cells under Com-Phe stress were analyzed by the RNA-seq technology,the results showed that Com-Phe altered some key physiological processes,including membrane transporters,primary carbon metabolism,antioxidative activity,sporulation,and cell division.Among all the differential expression genes,16 genes encoding ABC transporter were up-regulated,10 genes were down-regulated,and 4 genes related to glutathione metabolism were differentially expressed.Compared with the control,the key genes trxA(thioredox protein)and trxB(thioredox protein reductase)in this pathway were upregulated by 1.92-fold.In addition,genes related to spore formation and cell division also showed significant changes,and the ftsA gene encoding cell division protein was downregulated by 2.11-fold.In summary,the research results of this study would not only provide new insights for the optimization of pretreatment process of lignocellulosic biomass,but also provide theoretical foundation for constructing robust strains with high tolerance to the pretreatment-derived inhibitors by metabolic engineering approach.