Structural Elucidation Of Lignin From Biomass And Its Dissociative Mechanism During Pretreatment Process

Lignin is one of main components of Lignocellulosic biomass. In the past few decades, although many researchers in the wood chemistry and pulping chemistry devoted their endeavors to uncover lignin complex structures, two main questions regarding to its chemical structures should be deeply investigated. The first question is how to isolate lignin from biomass via “unaltered form”. Another question is how to select and develop more precise method to analysis the chemical structures of lignin. With the development of “biorefinery”, more questions regarding lignin should be illuminated, such as the dissociative mechanism of lignin during pretreatment. The solutions to the above-mentioned questions will not only promote the development of wood chemistry, but also provide some theoretical foundations to the value-added applications of the lignin. In this doctoral thesis, some aspects regarding to the isolation of “native lignin” from plant cell wall and its structures analysis were firstly presented. Based on the isolation and analytical methods presented, the fundamental chemistry of lignin and chemical transformations of lignin during different leading pretreatments will be also investigated. In addition, based on the results obtained, some feasible methods for disassembling the plant cell wall into main components will be presented in a biorefinery scenario.(1) In the first chapter of the doctoral thesis, modified milled wood lignins(MWL) were isolated from the stem(MWLS) and pith(MWLP) of bamboo(Phyllostachys pubescens). The modified MWL have some advantages, such as higher yield, lower content of carbohydrates, and typical structures. The non-acetylated and acetylated bamboo MWLs were investigated by FTIR, quantitative 13C-NMR, 2D-HSQC NMR, and 31P-NMR spectroscopy. The MWL consist of p-hydroxyphenyl(1~2%), guaiacyl(21~31%), and syringyl(67~78%) units associated with p-coumarates and ferulates. A modified quantitative 13C-NMR and 2D-HSQC analysis have demonstrated that the predominant intermonomeric linkages are of the type β-O-4(45~49 per 100 C9-units, i.e. per C900) along with small amounts of other structural units such as resinols(3.6~7.4 per C900), tetrahydrofuran(2.0~2.3 per C900), phenylcoumaran(2.8~4.5 per C900), spirodienones(1.3~2.3 per C900) and α, β-diaryl ethers(2.8~2.9 per C900). MWLP contained more p-coumarates than MWLS. The various degrees of γ-acylation(17~27%) were positively associated with S/G ratios in the lignins, however, γ-acylation was inversely correlated to the ratio between β-β and β-O-4 side-chains in the lignin fractions. Moreover, a flavonoid compound(tricin) was also detected in the MWLS but not in MWLP. The two MWLs are very similar in terms of molecular weights and the contents of OHphen and OHaliph.The heterogeneity of the lignin is reflected not only in different parts of a whole plant, but also in different fractions of a location of the plant. To reveal the heterogeneity of the lignin, a combined method based on sequential extractions was established in this study for analyzing structural features of native lignin from bamboo species. The chemical and structural inhomogeneity of the isolated native lignin samples were comparatively and comprehensively investigated by elemental analysis, FT-IR spectra, quantitative NMR spectra(13C-NMR, 13C-DEPT135, 2D-HSQC, and 31P-NMR), and GPC techniques. The C900 experiential formula of the lignin is C900H865O330(OCH3)133 and C900H815O320(OCH3)136 for MWL and AL, respectively. In addition, it was found that S/G and ether bonds(β-O-4 and α,β-ether bonds) are increased in the subsequently extracted AL, suggesting that MWL may mainly originated from middle lamella, and its structures are also affected by ball-milling process, while the AL are mianly derived from secondary cell wall S2 layer.Traditionally, the lignin used for structural analysis is usually based on extraction. In this study, a simple method based on DMSO/NMI(dimethylsulfoxide/N-methylimidazole) dissolved system for the isolation of dissolved lignin(DL) from bamboo was presented. The lignin extracted was studied as compared to milled wood lignin(MWL) and alkali lignin(AL). Quantitative 13 C and 2D-HSQC NMR analysis indicated that all lignin preparations are HGS-type and partially acylated(below 12%) at the γ-carbon of the side chain by p-coumarate. The abundances(Ar/100Ar) of β-O-4, β-β, and β-5, S/G ratios, acylation degree, phenolic hydroxyl, aliphatic hydroxyl and methoxy groups(mmol/g) were also observed in the lignin preparations. Based on the above-mentioned results, in-situ HSQC-NMR characterization was used to solve the following questions:(1) The effect of dissolved system(DMSO/NMI) on the structures of the residual lignin;(2) structural analysis of the residual enzyme lignin(REL). Specially, the NMR spectra were successfully obtained by dissolving the acetylated and non-acetylated bamboo samples in appropriate deuterated solvent(CDCl3 and DMSO-d6). The heterogeneous lignin polymers in bamboo samples were demonstrated to be HGS-type and partially acylated at the γ-carbon of the side chain by p-coumarate and acetate groups. In addition, it was found that the removal of DL enhance the S/G ratio of residual lignin. The S/G ratios are listed as following orders: RB<CEL<REL. Inspiringly, the method gives us a vision to track the structural changes of plant cell wall(e.g. lignin polymers) during the different pretreatment processes.A comprehensive understanding of the structures of whole lignin in the plant cell wall is extremely significant for developing effective biomass processing and utilization. In this study, a robust method based on mild alkaline preswollen and in-situ enzymatic hydrolysis for the isolation of “swollen residual enzyme lignin, SREL” from Eucalyptus wood was presented. The SREL obtained was investigated as compared to the corresponding cellulolytic enzyme lignin(CEL) and alkali lignin(AL). Remarkablely, the yield of SCEL(95%) was significantly higher than that of CEL(20%) and AL(12%). The isolated lignin polymers have been comparatively investigated by a combination of elemental analysis, 2D HSQC NMR, 31P-NMR, analytical pyrolysis, and GPC techniques. The major lignin linkages(β-O-4, β-β, and β-5, etc.) were thoroughly assigned and the frequencies of the major lignin linkages were quantitatively obtained. In particular, p-hydroxyphenyl(H) units were observed in SREL and AL rather than CEL, suggesting that H-type lignin units, which are difficult to be extracted with 96% dioxane, could be obtained by this method. Inspiringly, the effective method gives us an integrated vision to understand the intrinsic structural features of whole lignin from plant cell wall and helps to develop more effective plant deconstruction or pretreatment strategies in the current biorefinery process.Due to the drawbacks of the structural analysis of lignin during pretreatment, the dissociative mechanism of the lignin during the auto-catalyzed organosolv and ionic liquid pretreatments were thoroughly investigated based on the “Quantitative NMR Methods”. During auto-catalyzed organosolv pretreament, the chemical structural elucidation of the isolated birch lignins was performed using multiple NMR methodologies(31P-, 13C- and 2D-HSQC NMR techniques). Results showed that the amount of β-O-4 linkages decreased in the order of AEOL(auto-catalyzed ethanol organosolv lignin) < EHLP(enzymatic hydrolysis lignin, pretreated) < EHLU(unpretreated). The homolytic cleavage of β-O-4 linkages resulted in an increase of free phenolic hydroxyl groups and carboxylic acids in AEOL and EHLP as compared to that of EHLU. In addition, α-ethoxylation was the only modification in the auto-catalyzed ethanol organosolv pretreatment(AEOP). Another, to understand the structural changes of lignin after pretreatment and enzymatic hydrolysis process, ionic liquid lignin(ILL) and subsequent residual lignin(RL) were sequentially isolated from ball-milled birch wood. The quantitative structural features of ILL and RL were compared with the corresponding cellulolytic enzyme lignin(CEL) by nondestructive techniques(e.g. FTIR, GPC, Quantitative 13 C, 2D and 31P-NMR). The IL pretreatment caused structural modifications of lignin(cleavage of β-O-4 ether linkages and formation of condensed structures). In addition, lignin fragments with lower S/G ratios were initially extracted, while the subsequently extracted lignin is rich in syringyl unit. Moreover, the maximum decomposition temperature(TM) was increased in the order of ILL<RL<CEL, which was related to the corresponding β-O-4 ether linkages content and molecular weight(Mw). Based on the results observed, a possible separation mechanism of IL lignin was proposed.Unveiling the fundamental chemistry of lignin under ionic liquid(IL) pretreatment will facilitate the understanding of biomass recalcitrance involved in pretreatment processes. To examine in greater detail the chemical transformations of lignin under different IL pretreatment conditions without competing reactions from plant polysaccharides, the IL pretreatment of the isolated poplar alkaline lignin(hardwood lignin) under varying IL pretreatment conditions(i.e., 110-170 oC, 1-16 hour) was performed in an appropriate manner. The structural transformations of the lignin have been investigated by elemental analysis, 2D-HSQC spectra, quantitative 13C-NMR spectra, 31 P NMR, and GPC analysis. Results revealed that a decrease of aliphatic OH and an increase in phenolic hydroxyl groups occurred in lignin as the pretreatment proceeded. The increased phenolic OH was mainly as a result of cleavage of β-O-4 linkages, while the reduced aliphatic OH is probably attributed to the dehydration reaction. The cleavage of β-O-4 linkages, degradation of β-β and β-5 linkages obviously occured in high temperatures and resulted in the decrease of molecular weights. In addition, IL pretreatment selectively degraded G-type lignin fractions and condensation reaction more easily took place at S units than G units. Moreover, the demethoxylation preferentially occurred in G units, especially at higher temperatures. It is believed that investigating the fundamental chemistry of lignin during IL pretreatments would be beneficial to optimize and control the pretreatment process.Based on the understanding of lignin structures from bamboo and Eucalyptus wood, two effective isolation methods were developed. Bamboo(P. pubescens) was successfully fractionated using a three-step integrated process:(1) autohydrolysis pretreatment facilitating xylooligosaccharide(XOS) production(2) organosolv delignification with organic acids to obtain high-purity lignin, and(3) extended delignification with alkaline hydrogen peroxide(AHP) to produce purified pulp. The integrated process was comprehensively evaluated by component analysis, SEM, XRD, and CP-MAS NMR techniques. Emphatically, the fundamental chemistry of the lignin fragments obtained from the integrated process was thoroughly investigated by gel permeation chromatography and solution-state NMR techniques(quantitative 13 C, 2D-HSQC, and 31P-NMR spectroscopies). It is believed that the integrated process facilitate the production of XOS, high-purity lignin, and purified pulp. Moreover, the enhanced understanding of structural features and chemical reactivity of lignin polymers will maximize their utilizations in a future biorefinery industry. Eucalyptus chips were successively subjected to organosolv pretreatment and extended delignification process in the present study. The effects of delignification processes were scientifically evaluated by component analysis, SEM, and CP-MAS NMR techniques. The fundamental chemistry of the lignin polymers obtained after these processes were thoroughly investigated by FT-IR, multi-dimensional NMR(31P-, 13C- and 2D-HSQC NMR), and GPC techniques. Auto-catalyzed ethanol organosolv pretreatment(AEOP) process and extensive delignification(ED) resulted in an effective delignification. It was observed that an extensive cleavage of β-O-4 linkages, α-ethoxylation, transformation(stilbene unit), and some condensation reactions occurred in AEOP process, while α-oxidation mainly took place in alkaline hydrogen peroxide(AHP) process. It is believed that better understanding the fundamental chemistry of lignin would be beneficial to optimize and control the delignification processes. More importantly, well-characterization of lignin polymers will facilitate their value-added applications in current and future biorefineries.Torrefaction is an efficient method to recover energy from biomass. Herein, the characteristics(mass yield, energy yield, physical, and chemical characteristics) of torrefied bamboo at diverse temperatures(200-300 oC) were firstly evaluated by elemental analysis, XRD, and CP-MAS 13C-NMR methodologies. Under an optimal condition the terrified bamboo has a relative high energy yield of 85.7% and a HHV of 20.13 MJ/Kg. The chemical and structural transformations of lignin induced bythermal treatment were thoroughly investigated by FT-IR and a series of solution-state NMR techniques(quantitative 13C-NMR, 2D-HSQC, and 31P-NMR methodologies). The results highlighted the chemical reactions of the native bamboo lignins towards severe torrefaction treatments occurred, such as depolymerization, demethoxylation, bond cleavage, and condensation reactions. NMR results indicated that aryl-ether bonds(β-O-4) and p-coumaric ester in lignin were cleaved during the torrefaction process at mild conditions. The severe treatments of bamboo(275 oC and 300 oC) induced a dramatic enrichment in lignin content together with the almost complete disappearance of β-O-4, β-β, and β-5 linkages. Further analysis of the molecular weight of milled wood lignin(MWL) indicated that the average molecular weights of “torrefied MWL” were lower than those of control MWL. It is believed that understanding of the reactivity and chemical transformations of lignin during torrefaction will contribute to the integrated torrefaction mechanism.