Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides (original) (raw)
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Holzforschung, 2000
Brown rotted Scots pine (Pinus sylvestris) sapwood was studied using scanning UV microspectrophotometry. Wood blocks were exposed to the fungus Coniophora puteana (Schum.: Fr.) Karst. (BAM Ebw.15) for 6, 8, 10, 30, and 50 days. No wood weight loss was detected in the initial decay periods. On the other hand, point analyses of lignin distribution in wood cells revealed higher absorbance after 6-10 days of decay, which we interpret as an increase in the absorption coefficient of lignin due to its oxidative modification by the fungus. Uneven wood degradation occurred in the later periods (30 and 50 days), with both significantly decayed and visually sound cells observed. The decayed cells showed a higher absorbance at 280 nm, although the apparently sound cells were also degraded to a lower extent. Degradation of lignin-rich compounds in middle lamellae and cell corners was not observed during fungal attack.
Microscopy Research and Technique, 2013
Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO 4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production.
Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells
PLoS ONE, 2013
Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulosebinding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.
Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type
Scientific Reports
Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery. Plant cell walls constitute the largest renewable source of biomass on Earth that can supply environmental benefits for the production of fuel, chemicals and materials. They are composed by lignocellulose made from three main polymers (cellulose, hemicelluloses, lignin) assembling as a network whose structural and chemical complexity hampers hydrolysis of cellulose by enzymes and microorganisms 1. In addition, cell walls display high variability depending on genetic and environmental factors, as well as plant tissue and cell types 2-4. Understanding plant cell wall complexity during lignocellulosic bioconversion is therefore important to identify critical features impacting hydrolysis, for optimising pretreatments of biomass 5 and enzyme cocktails 6. Investigation of the dynamics of lignocellulose hydrolysis requires physicochemical characterization and multiscale visualization 7,8. Combined approach using multiple microscopic techniques including scanning electron microscopy, atomic force microscopy, light microscopy, immunocytochemistry and microspectrometry can be used to monitor microstructural and topochemical heterogeneity of plant cell walls and their recalcitrance at tissue, cell and subcellular levels 9-15. In addition, spatial and temporal imaging of enzymes distribution within lignocellulose substrates can be carried out by means of immunoprobe or fluorescent labelled protein 16-18 to study accessibility at the molecular scale 19,20. However real time imaging of cell wall microstructure and enzyme distribution during bioconversion still remains challenging. Enzymatic degradation of cellulose and hemicelluloses involves several types of enzymes, namely glycoside hydrolases that work synergistically 21,22. To overcome the recalcitrance of plant polysaccharides, filamentous fungi and bacteria secrete lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of glycoside bonds 23-25. In industrial processes, addition of LPMOs to cellulolytic cocktails leads to the reduction of the enzyme loading required for efficient saccharification of lignocellulosic biomass 26. These powerful enzymes are copper-containing enzymes classified within the auxiliary activity (AA) class of the CAZy database (www.cazy. org 27) in AA9-AA11 and AA13 families. AA9 LPMOs are mostly active on cellulose but some have been shown to act on xyloglucan and glucomannan 25,28. Despite their recognized boosting effect on biomass hydrolysis, AA9 LPMOs activity has been essentially investigated on model substrates with only sparse studies focusing on the insoluble fraction of the substrate that show their disruptive action at the surface of cellulosic fibers 29-31. To our
Journal of Agricultural and Food Chemistry, 2005
Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga-and Mä ule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.
Biotechnol …, 2010
Background: Lignin is embedded in the plant cell wall matrix, and impedes the enzymatic saccharification of lignocellulosic feedstocks. To investigate whether enzymatic digestibility of cell wall materials can be improved by altering the relative abundance of the two major lignin monomers, guaiacyl (G) and syringyl (S) subunits, we compared the degradability of cell wall material from wild-type Arabidopsis thaliana with a mutant line and a genetically modified line, the lignins of which are enriched in G and S subunits, respectively. Results: Arabidopsis tissue containing G-and S-rich lignins had the same saccharification performance as the wild type when subjected to enzyme hydrolysis without pretreatment. After a 24-hour incubation period, less than 30% of the total glucan was hydrolyzed. By contrast, when liquid hot water (LHW) pretreatment was included before enzyme hydrolysis, the S-lignin-rich tissue gave a much higher glucose yield than either the wild-type or G-ligninrich tissue. Applying a hot-water washing step after the pretreatment did not lead to a further increase in final glucose yield, but the initial hydrolytic rate was doubled. Conclusions: Our analyses using the model plant A. thaliana revealed that lignin composition affects the enzymatic digestibility of LHW pretreated plant material. Pretreatment is more effective in enhancing the saccharification of A. thaliana cell walls that contain S-rich lignin. Increasing lignin S monomer content through genetic engineering may be a promising approach to increase the efficiency and reduce the cost of biomass to biofuel conversion.
Sugarcane culm has different types of tissue organization, and its heterogeneity can influence the bagasse quality generated in the sugar/ethanol industry. The heterogeneity of the sugarcane bagasse contributes to the intrinsic recalcitrance of the biomass, impairing its conversion to ethanol. To determine the effects of sugarcane culm heterogeneity, the internode and node fractions were pretreated with acid, alkali, and peroxide. The investigation focused on the change in the fraction morphology, crystallinity, chemical composition, and enzymatic digestibility. Scanning electron microscopy (SEM) revealed different anatomical traits between the node and the internode. Vascular bundles appeared in larger number and diameter in the periphery of both the node and the internode. The internode and node responded differently to the pretreatments, with slight differences in the amount of hemicellulose and lignin removal. Furthermore, the internode was more susceptible to the acid and alkaline pretreatments than the node, generating a material with better digestibility. The fractions pretreated with peroxide showed similar enzymatic digestibility. In SEM images, the internode showed more structural damage after pretreat-ments than the node fibers. These results provide insight that pretreated sugarcane bagasse has fractions with different enzymatic digestibility related to their anatomy, and that conversion of the entire biomass is not feasible.
Lignin Analysis in Arabidopsis Thaliana Using the Photometer-Microscope Mpmsoo
Walls is an important quality factor in nurnerous industrial processes. Especially ongoing breeding programs are trying to alter liguin and, tberefore, reliable and bigbresolution analysis methods for lignin are essential. In this Paper, we report data on the applicability of UV microscopy for the analysis of lignin composition in Xylem cell wal.ls of Arabidopsis thaliana (Schmalwand, thale cress) Sterns. The lignin composition was determined in different Parts of the Xylem cell Walls by measuring the absorbance at wavelengths of 235 to 400 nm. The resulting spectra indicate considerable variability of lignin content and composition between the middle lamellae, the secondary wall of vessels and fibers. UV microscopy has proven to be higbly useful for quantitative lignin analysis in Arabidopsis Xylem tissues.