Study On Chemical Linkage Between Cellulose And Lignin In Hardwood By ²H/¹³C Dual Isotopic Tracer

In the plant fiber raw materials,due to the close connection between cellulose,hemicellulose and lignin,and the existence of lignin-carbohydrate complexes,it is difficult to achieve efficient separation of cellulose.Therefore,deligninfication become difficult in the process of pulp and paper industry,viscose cellulose industry and biorefining.Many researchers have suggested that there are chemical connections between lignin and carbohydrate,such as benzyl ether linkage,benzyl ester linkage,and ketal linkage.But there are few studies on the chemical connection between lignin and cellulose.In order to remove lignin more thoroughly,it is necessary to explore the chemical connection between lignin and cellulose.In this paper,sodium acetate-1-¹³C and malonic acid-1,3-¹³C₂ were used as starting materials to synthesize lignin precursor coniferin-[?-¹³C]and coniferin-[?-¹³C].The chemical structure of intermediates products and target products were confirmed by FT-IR,micro melting point analyzer and ¹³C-NMR.Then,cellulose precursors uridine diphospho-glucose-6,6-D₂ and lignin precursors coniferin-[?-¹³C]and coniferin-[?-¹³C]were put into the oleander shoots during their growth period,then cultured in a artificial climate chamber for 30 days.We could find that there was no effect on the normal growth of the oleander shoots.The elemental analyzer and stable isotope ratio mass spectrometer were used to analyze the ²H and ¹³C abundances of the oleander newly-formed xylem.It was found that the ²H and ¹³C abundances were significantly higher than the natural plant.The results indicated that the cellulose and lignin in the oleander shoots had been labeled by ²H and ¹³C isotope successful.Isotope labeled precursors were mainly absorbed and metabolized in the primary wall layer,middle lamella layer and secondary wall layer of the cells.The high resolution solid nuclear magnetic CP/MAS ¹³C-NMR analysis of the oleander newly-formed xylem indicated that lignin sbustructural units were?-O-4,?-5,?-?,and?-1 etc.With ketal linkage and benzyl ether linkage between carbohydrate and??-C?lignin.Lignin-carbohydrate complexes?LCC?and milled wood lignin?MWL?were extracted from ²H/¹³C double isotope labeled oleander newly-formed xylem by the method of Bj?rkman.The ¹³C-NMR spectra analyses of MWL-[?-¹³C],MWL-[?-¹³C]and intact MWL of oleander showed there were?-O-4,?-5,?-?,?-1 and substructures in the MWL of oleander.¹³C-NMR and ¹H-NMR analysis results revealed that the bond between carbohydrate and lignin were benzyl ether linkage,benzyl ester linkage,and ester linkage at?-C of lignin side chain.In particular,the benzyl ether linkage between the C_?position of lignin and the glucose unit C₆ of cellulose was elucidated.In order to reduce the signal of sugar units which has no chemical bond connection with lignin as far as possible,in this work,the cellulase and hemicellulase was used to hydrolyze the lignin-carbohydrate complex.C_?-¹³C/6,6-D₂ dual-labeled LCC was enzymatic degraded with cellulase and hemicellulase to obtain dual-labeled Enzyme-degraded LCC?EDLCC?.The labeled EDLCCs were detected by ¹³C-NMR and ¹H-NMR.The results further confirmed that a small amount of ferulic acid and p-coumaric acid in the lignin structure could form an ester bond with the OH at the C₆ position of cellulose.In order to study the chemical structure of lignin and lignin-carbohydrate complexes more efficiently,cambium tissues were scraped from the oleander shoots during the growth period.The coniferin-[?-¹³C]were injected into the oleander cambium tissues during suspension culture of the cells.The ¹³C-enriched cell wall-dehydrogenated polymer?CW-DHP?was produced by the action of?-glucosidase,glucose oxidase,laccase,and manganese peroxidase in plant cells.As compared with?-¹³C labeled oleander plants,the preparation time of CW-DHP is shorter and the labeling effect is more obvious.As a substitute for ¹³C-enriched native plants,it is proposed a simpler technology to label native plants in situ.The CP/MAS ¹³C-NMR analysis of the oleander cambium tissues indicated that lignin substructures were?-O-4,?-5,?-?,and?-1 etc.Moreover,the bond between carbohydrate and?-C of lignin was proved to be ketal linkage,benzyl ether linkage and ester linkage.