| Biorefinery of lignocellulosic materials plays an important role of in ensuring energy security,protecting the environment and promoting the sustainable development of the society.However,high enzyme dosage caused by the recalcitrance of lignocellulosic materials results in high cost of production,and thus hinders the industrialization of the biorefinery.Enzymatic hydrolysis of lignocellulose was affected by lignin and phenolic compounds derived from the pretreatment process.In this study,the effects of lignin(derived from enzymatic hydrolysis of lignocellulosic biomass)and phenols on enzymatic hydrolysis and their interaction with enzymes were investigated.Based on above results and the kinetic study of enzymatic hydrolysis of dilute alkali and dilute acid pretreated corn stover,an enzyme recycling strategy was developed and the enzyme dosage was reduced via recycling un-hydrolyzed solids to next round fresh substrate.The main work includes:(1)Dilute alkali and dilute acid pretreatment was performed on corn stover and the pretreated corn stover was excessively enzymatic hydrolysised to obtain cellulolytic enzyme lignin(CEL).The adsorption profile of cellulase cocktail,Cellobiohydrolyase I(CBHI)and Endoglucanase I(EGI)on CEL was determined and the effect of CEL on enzymatic hydrolysis of Avicel was examined.The adsorption profile of cellulase onto CEL was investigated under different ionic strength,pH and urea concentrations.The results showed that CEL derived from dilute acid pretreated corn stover(CEL-acid-CS)generally absorbed more enzyme than CEL derived from dilute alkali pretreated corn stover(CEL-alkali-CS).The maximum adsorption capacity of cellulase cocktail on CEL-acid-CS and CEL-alkali-CS was 19.90 mg protein/g lignin and 10.09 mg protein/g lignin,respectively.Both CEL-acid-CS and CEL-alkali-CS had no significant effect on hydrolysis of Avicel.Elevated ionic strength,pH and urea concentration reduced the adsorption of cellulase onto CEL.(2)Enzymatic hydrolysis,enzyme adsorption and inhibitory effect of phenols was investigated on cellulose ?,cellulose ?,cellulose ? and amorphous cellulose.The correlation of cellulose crystallinity and inhibitory effect of phenols was also determined.Furthermore,the reduction of core enzyme activities and precipitation of enzymes resulted by phenols was studied.The results showed that the 72 h sugar conversion of cellulose I,cellulose II,cellulose III and amorphous cellulose was 80.2%,84.0%,93.3% and 100.0%,respectively.Cellulose easier to hydrolyze was less affected by phenols.With the concentration of phosphoric acid used to treat cellulose I increased from 0%(no treatment)to 84%,the sugar conversion at 72 h increased from 71.8% to 100.0%,and the inhibitory effect by phenols decreased from 62.2% to 4.8%,indicating that cellulose with lower crystallinity was less affected by phenols.Phenols lowered the CBH,EG,?G activities and caused precipitation.CBH activity was reduced by 57.4% and CBHs in cellulase cocktail almost precipitated completely in 72 h with 10 g/L vanillin.Both activity reduction and degree of precipitation of CBH was greater than EG and ?G.(3)Enzymatic hydrolysis and enzyme adsorption profiles of dilute alkali and dilute acid pretreated corn stover was studied and an enzyme recycling strategy via recycling unhydrolyzed solids after 24 h was developed.Both dilute alkali and dilute acid pretreated corn stover relased most sugar in the first 24 h,after which the sugar conversion only increased slightly.Dilute alkali pretreated corn stover converted 76.4% glucan and 56.0% xylan,absorbed 39.5% enzymes at 24 h and at the same time point,dilute acid pretreated corn stover converted 69.5% glucan and absorbed 54.9% enzymes.Enzyme recycling via recycling unhydrolyzed solids after 24 h shortened the hydrolysis time of each round to only 24 h and reduced the enzyme loading by 40% and 50% on dilute alkali and dilute acid pretreated corn stover,respectively,without reducing the sugar concentration in each round of enzymatic hydrolysis. |
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