Effects Of Lignin Structure On Cellulase Adsorption And Enzymatic Hydrolysis

Lignin is one of the important components in the cell wall of lignocellulosic biomass.It gives plant cell wall rigidity and strength and protects them from microbial attack.The complexity of lignin structure makes it difficult to determine the mechanism of lignin on cellulase adsorption and cellulase hydrolysis.Taking bamboo,poplar and masson pine(poplar,eucalyptus and acacia)as raw materials,milled wood lignin(MWL)was separated by Bj?kman method.The effects of different structural unit types(contents)of lignin on cellulase adsorption and enzymatic hydrolysis were studied,and the data were fitted to establish a kinetic model,so as to establish the relationship between lignin structural units and cellulase.In order to better explain the effect of lignin structure on cellulase hydrolysis and improve the adsorption of cellulase and cellulose,hydroxypropyl sulfonylmethylation of lignin was modified by two-step method,and its effect on cellulase hydrolysis was explored.The main research contents and results are as follows:1.Milled wood lignin was extracted from bamboo,poplar and masson pine by Bj?kman method.The lignin samples were characterized by elemental analysis,gel permeation chromatography,nitrobenzene oxidation and infrared spectroscopy.The results showed that bamboo lignin was composed of three structural units:syringyl,guaiacyl and p-hydroxyphenyl,and the S/G ratio was 1.83;Poplar lignin was composed of lilac based and guaiac based structural units,S/G=0.98;However,masson pine lignin only contains guaiac based structural units.The effect of lignin structural units on enzymatic hydrolysis was studied by dissipative quartz crystal microbalance(QCM-D).According to the results of QCM-D,the affinity of masson pine lignin for cellulase was lower than that of poplar and bamboo lignin,and the adsorption capacity of lignin of different structural unit types for cellulase was also different.Compared with grass and hardwood lignin,softwood had weaker adsorption capacity for cellulase.2.In order to further study the effect of lignin structural units on cellulase adsorption and enzymatic hydrolysis,three different broad-leaved wood,poplar,eucalyptus and acacia,were selected as raw materials,and MWL was extracted after ball milling.Elemental analysis,gel permeation chromatography,nitrobenzene oxidation and infrared spectroscopy showed that the content and molecular weight of the three MWL were almost the same.Poplar,eucalyptus and acacia MWL were GS lignin,and the main connection mode between structural units wasβ-O-4 structure,which also contains a small amount ofβ-5、β-β.Their S/G ratios were 0.99,1.92 and 1.34 respectively.The adsorption results of Cellulase on the surface of three kinds of separated lignin films showed that due to the higher S/G ratio of eucalyptus MWL,more cellulase was coated in the network structure of lignin.Compared with the other two MWLs,eucalyptus MWL had stronger affinity for cellulase.The lignin film adsorbed with cellulase was characterized by atomic force microscope(AFM).The cellulase was evenly distributed on the surface of the film,resulting in the reduction of the roughness of the film.Because of its strong affinity for cellulase,eucalyptus MWL made the roughness of lignin film after adsorption higher than poplar and acacia MWL.Based on the QCM dynamic parameters of enzymatic hydrolysis process of lignin/cellulose composite film,the enzymatic hydrolysis process of composite film can be divided into four stages:adsorption,rapid hydrolysis,slow hydrolysis and hydrolysis equilibrium.Combined with the kinetic parameters of enzymatic hydrolysis,eucalyptus lignin with higher S/G ratio takes longer time to reach the maximum enzymatic hydrolysis conversion and lower hydrolysis degree.Similarly,the hydrolyzed film was characterized by AFM.After cellulase hydrolysis,more lignin originally coated was exposed and showed irregular structure,which increased the roughness of the composite film.The higher the degree of hydrolysis,the greater the roughness of the composite film.3.Hydroxypropyl sulfomethylation of lignin was modified by two-step method,and the structure and surface properties of modified lignin were characterized by IR,~1H NMR,surface charge analysis and contact angle analysis.IR and ~1H NMR spectra showed that phenolic hydroxyl groups in lignin were blocked and sulfonic acid groups were introduced.The analysis of surface charge and contact angle showed that compared with lignin,lignin after hydroxypropyl sulfonylmethylation had higher electronegativity and lower hydrophobicity.The adsorption capacity of hydroxypropyl sulfomethylated lignin on cellulase was investigated by dissipative quartz crystal microbalance.The results showed that the force between modified lignin and cellulase was weakened and the ineffective adsorption was reduced.Taking microcrystalline cellulose and microcrystalline cellulose added with lignin or hydroxypropyl sulfomethylated lignin as substrates,the effect of lignin on enzymatic hydrolysis was studied under the conditions of different enzyme dosage and different solid-liquid ratio.The experimental results further proved that hydroxypropyl sulfomethylated lignin had higher negative charge and lower hydrophobicity,and its inhibition on enzymatic hydrolysis was weakened,which could further improve the conversion efficiency of enzymatic hydrolysis.The microcrystalline cellulose was also modified in this study.After element analysis,infrared spectroscopy and X-ray diffraction,it was found that the element content of the treated microcrystalline cellulose was basically the same as that before treatment,and still retained the typical structure of cellulose.The results of X-ray diffraction showed that the crystallinity of microcrystalline cellulose decreased after hydroxypropyl sulfomethyl treatment,which was expected to promote the adsorption of cellulose by cellulase and improve the hydrolysis and conversion rate of cellulase.