The Study Of Organic Solvent Pretreatment To Preparation Of Fermentable Sugar And Co-production Of Levulinic Acid Of Corn Stover

Corn stover has the characteristics of large yield,low cost,and renewable,which is a potentially material for bioethanol production.However,the recalcitrant cellular structure hinders the hydrolysis and saccharification efficiency of cellulase.In this paper,aromatic sulfonic acid(p-TsOH)and organic solvent ethanol were used to pretreat corn stover to destroy the dense structure of cell wall,remove lignin and hemicellulose,and improve the hydrolysis and saccharification efficiency of cellulase,and was compared to ethanol/water prereatment.The hydrolysate after p-TsOH/water pretreatment can be reused.After repeated recycling,more hemicellulose hydrolysates(xylose and glucose)are released.Under the catalysis of p-TsOH/water,they are converted into high-value levulinic acid platform compounds by simple heating treatment.Based on the study of the mechanism of the ineffective adsorption of residual lignin on cellulase,we used polyethylene glycol compounds to chemically modify cellulase,which reduced the ineffective adsorption of residual lignin on cellulase and improved the hydrolysis efficiency of cellulase.The results showed that:After p-TsOH/water and ethanol/water pretreatments,the lignin content of corn straw significantly decreased.About 83.4%of the lignin was removed,after p-TsOH/water pretreatment at a relatively low temperature of 100 ~oC.However,the lignin removal rate after ethanol/water pretreatment at 220 ~oC was 67%.Under the conditions of cellulase dosage of 15 FPU/g-cellulose and substrate concentration of5%,the glucose yield of p-TsOH/water pretreated corn stover was 97.6%after 96 h hydrolysis,which was higher than that of 89.5%after ethanol/water pretreatment.When the cellulase dosage was reduced to 7.5 FPU/g-cellulose,the glucose yield after p-TsOH/water pretreatment was still higher than 80%,and that after ethanol/water pretreatment was only 65.2%.The composition and structure analysis of corn stover before and after pretreatment showed that p-TsOH/water pretreatment seriously damaged the fiber structure of corn stover,removed more lignin and hemicellulose,improved the accessibility of cellulose,resulting in a higher glucose yield.The prehydrolyzed solution was reused for 4 times to pretreat corn stover under the optimal p-TsOH/water pretreatment condition(100 ~oC,90%p-TsOH).When the cellulase dosage was 15 FPU/g-cellulose and the substrate concentration was 5%,the highest glucose yield of 98.9%was obtained after 2 times prehyfrolysate recycling.The study of lignin adsorptiosion showed that residual lignin from untreated corn stover had stronger cellulase adsorption capacity than residual lignin from pretreated corn stover.The cellulase after lignin adsorption partially lost its activity,and the structure of the adsorbed cellulase was characterized.The results show that the peak positions and maximum emission wavelengths corresponding to amino acid residues have different degrees of red shift.The greater the degree of red shift,the stronger the adsorption of residual lignin to cellulase.SDS-PAGE analysis showed that BGL and CBH in cellulase were more easily adsorbed by residual lignin.In addition,analysis of contact angle,Zeta potential,FT-IR and XPS on residual lignin showed that increasing the hydrophilicity of lignin and reducing the electronegativity of residual lignin could reduce the ineffective adsorption of lignin on cellulase.Polyethylene glycol(PEG),hydroxy-polyethylene glycol-carboxyl(OH-PEG-COOH),and carboxy-polyethylene glycol-carboxyl(COOH-PEG-COOH)with different molecular weights activated by melamine are modifying agents to mnodify the cellulase.The results showed that the enzyme activity of the filter paper of the modified enzyme was higher than that of the natural cellulase.Among them,the highest activity of 92.7 FPU/mL was reached after COOH-PEG-COOH-5Kmodification.The modified enzymes were characterized by ultraviolet spectroscopy,fluorescence spectroscopy,circular dichroism chromatography and SDS-PAGE.The results showed that the COOH-PEG-COOH-5K hase the best modification effect on increasing the outward exposure of tryptophan residues,decreasing the outward exposure of tyrosine and phenylalanine residues,and fluorescence.By modifying the circular dichroism spectrum of the enzyme,the secondary conformation structure of the protein was also changed.COOH-PEG-COOH-5K,which has the best modification effect,reduced the?-helix of cellulase and improved the?-sheet of cellulase.During cellulase hydrolysis reaction of natural enzymes and modified enzymes,and adsorption reaction of lignin with cellulase before and after modification,it is clear that modified enzymes reduced the adsorption capacity on lignin,and increased the enzymatic hydrolysis.More monomeric sugars(xylose and glucose)derived from hemicellulose were released in prekysrolydate after p-TsOH/water pretreatment.They could be converted to valuable platform chemical of levulinic acid by simply heating the prehydrolysate.The effects of reaction time and reaction temperature on the reaction results were investigated by using the pre-hydrolyzed solution 4 times after removing lignin as raw materials.It was found tant the maximum levulinic acid yield was 58.1%at a reaction temperature of 190 ~oC and a reaction time of 1 h was obtained.Methyl isobutyl ketone(MIBK)was used to extract the levulinic acid after the reaction to obtain a relatively pure levulinic acid product.The FT-IR and ~1H NMR analysis of the product showed that the levulinic acid product contained a small amount of p-TsOH,and p-TsOH was recovered by crystallization technology.