【作者】 马建锋；

【导师】 许凤；

【作者基本信息】 北京林业大学 ， 林产化学加工工程， 2014， 博士

【摘要】 木质纤维生物质细胞壁微观结构复杂性及主要组分(纤维素、半纤维素以及木质素)分布不均一性构成了生物质细胞壁主要的天然抗降解屏障(Natural Biomass Recalcitrance)。为了提高生物质转化效率,降低转化成本,选择经济、高效、环保的预处理方法非常重要,它能有效地打破细胞壁抗降解屏障,增加酶对生物质多糖的可及性。本研究利用显微光谱技术阐明了木质纤维生物质发育过程中组分积累过程,同时结合显微及显微光谱技术在多尺度下解译了木质纤维生物质细胞壁组分微区分布及超微结构特点,在以上研究基础上在细胞水平探索了化学预处理过程中生物质细胞壁微观结构变化及组分溶解规律。主要结论如下：1.毛白杨(Populus tomentosd)形成层发育过程中木质部纤维细胞碳水化合物的积累先于木质素；而木质素的积累开始于细胞角隅区；相比于前一年的晚材纤维细胞,新形成的纤维细胞次生壁中纤维素浓度低于前一年的晚材纤维细胞,而木质素的浓度呈相反趋势2.黑杨(Populus nigra)纤维细胞壁分为：胞间层(ML)、初生壁(P)以及次生壁(次生壁外层S1,次生壁中层S2和次生壁内层S3),且各形态区域中木质素分布具有明显不均一性。除此之外,在S2层中出现电子密度较低的条纹状区域,这些区域表明纤维素微纤丝取向的空间取向差异3.黑杨(Populus nigra)受拉木(TW)纤维细胞出现额外的凝胶层(GL),且这一层具有较高纤维素浓度及痕量木质素；相比于对应木(OW)次生壁(Sw), TW的GL层中具有更为丰富的空隙结构；另外,TW纤维细胞Sw以及GL中负的拉曼特征峰(1097cm-1)位移表明TW形成过程中这两层产生了永久的拉伸形变4.连翘(Forsythis suspensa)形态学和解剖学的特点表明其具有替代传统林产品原料的潜在价值。其细胞壁木质素分布不均一性不仅存在于不同细胞间,也存在于同一细胞不同形态区域内。半定量的扫描电子显微镜结合能谱(SEM-EDXA)分析结果表明细胞角隅胞间层(CCML).复合胞间层(CML)、次生壁S2层的木质素浓度比为1.3：1.1：15.红瑞木(Cornus alba)茎部组织细胞中木质素和纤维素在不同细胞同一形态区域以及同一细胞不同形态区域的分布呈现明显的不均一性；木质素生物合成的前驱物质松伯醇和松柏醛(Lignin-CAA)汇聚于富含纤维素的次生壁中。另外,研究发现纤维细胞间的纹孔膜区域富含木质素及少量的纤维素及果胶类物质6.芒草(Miscanthus sinensis)节间组织厚壁纤维细胞(Sf)的分层特点具有明显的区域性；节间组织初生木质部导管(Pxv)和次生木质部导管(Mxv)次生壁的分层Sf简单；节间组织Pxv由微纤丝无序排列的初生壁(P)以及微纤丝水平排列且环状增厚的次生壁(Sw)组成7.芒草(Miscanthus sinensis)节间组织中木质素主要聚集在后生木质部导管(Mxv)、表皮细胞(Epi)、厚壁纤维细胞(Sf)次生壁及胞间层区域(ML)；而对羟基肉桂酸主要存在于Sf次生壁、薄壁细胞(Par)、Mxv以及ML；在亚细胞水平对羟基肉桂酸浓度与木质素浓度存在明显伴生关系8.离子液体1-乙基-3-甲基咪唑醋酸盐([Emim][OAc])预处理造成了虎皮松(Pinus bungeana Zucc.)对应木(OW)管胞次生壁明显的润胀,而应压木(CW)管胞润胀不明显；在oW管胞中,离子液体主要经细胞腔渗透到次生壁的内侧,进而扩散到次生壁临近复合胞间层的区域中。相比而言,离子液体在CW管胞中较难渗透；预处理后oW次生壁临近复合胞间层的区域中,碳水化合物、纤维素和木质素的变化程度大于其他形态区域,而CW管胞组分几乎没有变化更多还原

【Abstract】 Lignocellulosic plant cell walls are composed of crystalline cellulose nanofibrils embedded in an amorphous matrix of cross-linked lignin and hemicelluloses that impedes enzyme and microbial accessibility. The complex cell wall micro-structure and heterogeneous distribution of cell wall components are believed to be the natural biomass recalcitrance. In order to increase overall process efficiency and reduce costs, pretreatment of biomass is necessary to improve the accessibility of biomass polysaccharides to enzymatic hydrolysis. While various pretreatments have been employed, a detailed cellular level understanding of the pretreatment process is lacking due in part to complexity of the biomass composition and structure as well as interference from traditional processing chemicals that sometimes alter the material under study. In the present work, microscopic and microspectroscopic techniques were used to investigate cell wall micro-structural and topochemical information at cellular and sub-cellular. The conclusions obtained were as follows:In the developing xylem tissue of Populus tomentosa, prior to fiber lignification, the cellulosic polysaccharides have been deposited. The fiber lignification started from cell corner middle lamella (CCML), and then extends into secondary wall (Sw), The lignification level for the newly formed fiber Sw was higher than that of the previous latewood fiber.TEM images exhibited that the Populus nigra fiber wall was typically differentiated into three layers:middle lamella (ML), primary wall (P) and secondary wall (S1, S2and S3), and the staining intensities represented differing lignin concentrations. The striated appearance in the fiber S2indicated the orientation of cellulose microfibirls.Compared to Populus nigra opposite wood (OW), the tension wood (TW) fiber displayed an additional gelatinous layer (GL) with higher cellulose and less lignin. Meanwhile, the cellulose enriched GL in TW had much more abundant porosity than that of OW. Moreover, the microfibrils in the TW fiber S2and GL were stretched during TW formation and the microfibrils still keep the tensional deformation even after the fibers are transversally cut.Anatomical observations indicated that Forsythia suspensa was diffuse-porous wood. Helical thickenings and alternate intervessel pits were present on vessel cell wall. Confocal images (488nm) revealed a high level of lignin autofluorescence in the cell corner middle lamella (CCML), with lower levels of fluorescence in the compound middle lamella (CML) and S2region. The results from SEM-EDXA demonstrated that lignin concentration ratio in different regions of fibre wall was1.3(CCML):1.1(CML):1(S2).The inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers was observed in Cornus Alba. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber ceJl wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall (Sw) where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose and pectin was visualized in the pit membrane (PM) areas between fibers.A great degree of inhomogeneity in the layering structure of Miscanthus sinensis sclerenchymatic fiber (Sf) secondary wall (Sw) was visualized, while the Sw of xylem vessel can not clearly be divided into sub-layers. Moreover, we proposed an architectural model of protoxylem vessel (Pxv) composed of two layers:an outermost Pw composed of a meshwork of Mfs, and inner Sw containing regularly parallel Mfs.In herbaceous biomass Miscanthus sinensis lignin mainly accumulated within the secondary wall of epidermis, metaxylem vessel, sclrenchyma fiber (Sf) and middle lamella (ML). And higher concentration of hydroxycinnamic acids (HCA) was located at the secondary wall of Sf, parenchyma (Par), metaxylem vessel (Mxv) and ML. Moreover, a clear accompanied trend between lignin and HCA distribution within morphologically distinct cell wall layers of Sf and Par was observed.During ionic liquids ([Emim][OAc]) pretreatment of Pinus bungeana Zucc. opposite wood (OW), swelling occurred primarily in the secondary wall (Sw) adjacent to compound middle lamella (CML) and the ILs had little effect on the Sw adjacent to cell corner middle lamella (CCML). The time series of lignin Raman images showed that lignin decreased significantly from the Sw adjacent to Cml in OW while for the Sw of the CW tracheids there was no significant change of the lignin signal intensity within the same pretreatment time. Moreover, after washing with distilled water (80℃) OW tracheids displayed obvious decrease in lignin, carbohydrates and cellulose concentration.更多还原