Cellulose microfibril aggregates and their size variation with cell wall type

The organization of wood cell wall components involves aggregates of cellulose microfibrils and matrix known as macrofibrils. A combination of field emission electron microscopy and environmental scanning electron microscopy was used to visualise the organization of macrofibrils in different cell wall types comparing normal and reaction wood of radiata pine and poplar as examples of a typical softwood and hardwood. The size of macrofibrils is shown to vary among cell wall types with the smallest structures occurring in the gelatinous layer of tension wood (14 nm) and the largest structures in the S2_L layer of compression wood (23 nm). A positive correlation between macrofibril size and degree of lignification is observed, with macrofibrils apparently increasing in size in more highly lignified cell wall types. The fibrillar structure of the secondary wall varies from microfibril-sized structures of 3–4 nm up to large aggregates of 60 nm diameter. The size of macrofibrils also varies slightly among adjacent cells of the same cell wall type. Macrofibrils occur predominantly in a random arrangement, although radial and tangential lamellae may sometimes be seen in individual cells.

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In situ measurement of tensile elastic moduli of individual component polymers with a 3D assembly mode in wood cell walls

We attempted to measure in situ the tensile elastic moduli of individual component polymers with a three-dimensional (3D) assembly mode in the cell walls of Sugi (Cryptomeria japonica D. Don) without isolating the polymers. To prepare wood tangential slices [50 × 6 × 0.2 mm (L × T × R)] consisting of lignin with a 3D assembly mode in the cell walls, cellulose and hemicellulose were removed using the method of Terashima and Yoshida (2006) to obtain methylated periodate lignin slices. To prepare wood slices consisting of polysaccharide with a 3D assembly mode in the cell walls, lignin was removed using the method of Maekawa and Koshijima (1983) to obtain holocellulose slices. Static tensile test was applied to determine the elastic moduli of 3D lignin and 3D polysaccharide slices. The followings were revealed. The elastic modulus of the 3D lignin slices was 2.8 GPa, regardless of the microfibril angle (MFA) in the slices. The elastic moduli of the 3D polysaccharide slices with MFAs of 14°, 23°, 34°, and 42° were 18, 12, 9, and 4 GPa, respectively. The former shows that the lignin with a 3D assembly mode behaves as an isotropic substance in the cell walls, while the latter suggests that the 3D polysaccharide slice shows marked anisotropic structure in the cell wall. Despite the fact that cellulose content increased after lignin removal, values of substantial elastic modulus of the cell wall slightly decreased regardless of MFA. Following two possible reasons were pointed out for explaining this phenomenon. First, lignin removal caused an artifactual deterioration in the polysaccharide slices at the level of macromolecular aggregate. Second, rigid and fusiform-shaped cellulose crystallites are dispersed in the soft matrix of amorphous polysaccharide, and those are loosely connected to each other by the intermediary of matrix polysaccharide. Those suggest that the rigid cellulose crystallite can optimize its strong mechanical performance in the polysaccharide framework of the wood cell wall in combination with the ligninification.     

Fine structure of cellulose microfibrils in poplar gelatinous layer and Valonia

Summary Fine structure of cellulose microfibrils in poplar (Populus euramericana) gelatious layer and Valonia cell walls was observed in the electron microscope by using disintegration and ultrathin section techniques with various electron stains. Staining of the gelatinous layer in poplar showed that the microfibrils had a paracrystalline region surrounding a crystalline core, but such a region was negligible in Valonia. From the facts that kinks and shortened microfibrils were observed after mechanical and hydrolysis treatments, respectively, and that the microfibrils were unaffected by negative staining, it was concluded that the microfibril core is uniformly crystalline in the longitudinal direction although there may be some crystalline dislocations.The authors are indebted to Dr. Y. Yokohama (Marine Biological Laboratory, Tokyo University of Education) for supplying the samples and to Mr. Y. Tachida (Sanyo-Kokusaku Pulp Co.) for the technical assistance of the glow discharge apparatus.     

Nanostructural assembly of cellulose,hemicellulose, and lignin in the middle layer of secondary wall of ginkgo tracheid

Physical, chemical, and biological properties of wood depend largely on the properties of cellulose, noncellulosic polysaccharides, and lignin, and their assembly mode in the cell wall. Information on the assembly mode in the main part of the ginkgo tracheid wall (middle layer of secondary wall, S2) was drawn from the combined results obtained by physical and chemical analyses of the mechanically isolated S2 and by observation under scanning electron microscopy. A schematic model was tentatively proposed as a basic assembly mode of cell wall polymers in the softwood tracheid as follows: a bundle of cellulose microfibrils (CMFs) consisting of about 430 cellulose chains is surrounded by bead-like tubular hemicellulose-lignin modules (HLM), which keep the CMF bundles equidistant from each other. The length of one tubular module along the CMF bundle is about 16 ± 2 nm, and the thickness at its side is about 3–4 nm. In S2, hemicelluloses are distributed in a longitudinal direction along the CMF bundle and in tangential and radial directions perpendicular to the CMF bundle so that they are aligned in the lamellae of tangential and radial directions with regard to the cell wall. One HLM contains about 7000 C₆-C₃ units of lignin, and 4000 hexose and 2000 pentose units of hemicellulose.     

Enhancement of saccharification by overexpression of poplar cellulase in sengon

Lignocellulosic material from trees has great potential to form the basis of the second generation for bioethanol production because trees produce most of the biomass on the earth. We modified the wall structure of sengon (Paraserianthes falcataria) through overexpression of poplar cellulase in the cell walls. The overexpression did not decrease cellulose content but caused a decrease in xyloglucan bound to the walls. The level of saccharification and successive ethanol production was increased in the wood of the transgenic sengon overexpressing poplar cellulase compared with that of the wild type plant, and even after delignification of the wood. We propose that a xyloglucan intercalated into cellulose microfibrils could be one of the recalcitrant components in the saccharification of lignocelluloses.     

Mechanical constraints on lignin deposition during lignification

Summary The ultrastructure of lignifying cell walls in Pinus radiata D.Don was investigated using potassium permanganate staining and transmission electron microscopy. Lignin deposition occurred at numerous discrete sites within various cell wall regions, suggesting the presence of some initiating agent at these sites. In the middle lamella region, lignin deposition occurred by addition of protolignin monomers to spherical particles of lignin. Lignification was completed by expansion of these spherical particles, initially forming irregular patterns of lignification followed by infilling of adjacent areas. In contrast, lignification in the secondary wall occurred by deposition of protolignin monomers onto the ends of expanding lignin lamellae between cellulose microfibrils leading to greatly elongated patches of lignin due to the greater rate of deposition along the microfibril axis compared to that across it. It is concluded that the cellulose matrix in which lignin deposition occurs, in the secondary wall, can exert a mechanical influence which limits the rate of lignin deposition in the direction perpendicular to the microfibril axis.     

Microdistribution of metal elements in wood impregnated with a copper-chrome-arsenic preservative as determined by analytical electron microscopy

Summary The distribution of Cu, Cr and As in the walls of tracheids of Scots pine impregnated with Tanalith C at a retention of 2.5 lb/ft³ has been examined at the submicroscopic level in the electron microscope microanalyser, EMMA-4, using the absolute method, as well as in the conventional electron microscope. All three elements are present in all regions examined (probe area 0.2 m diameter). The concentration of Cu ranges from 0.4–0.9% (w/w), that of Cr is about 0.8%, while As ranges from 2%–2.6%. The morphology of this fine deposit is clearly dictated by the run of the cellulose microfibrils. There is in the wall an occasional coarse deposit, almost entirely copper, and the inner face of the tracheid wall is covered by a thin layer 20–30 m thick which contains all these elements at high concentration.     

Change in mechanical interaction between cellulose microfibril and matrix substance in wood cell wall induced by hygrothermal treatment

To investigate in detail the mechanical interactions and associations between cellulose microfibrils (CMFs) and the matrix substance, we measured the dimensional changes in cellulose crystals in wood cell walls after different treatments. The transverse expansion of CMFs observed after hygrothermal treatment and subsequent drying suggests that the matrix substance compresses the CMFs transversely under green conditions. However, as heat treatment breaks or weakens the association of the CMFs and the matrix substance, under hygrothermal treatment and drying at high temperature the matrix substance cannot compress the CMFs in the direction of the chain.     

Arrangement of cortical microtubules in compression wood tracheids of Taxus cuspidata visualized by confocal laser microscopy

Cortical microtubules (MTs) in differentiating compression wood tracheids of Taxus cuspidata stems were visualized by confocal laser microscopy. They were oriented obliquely at an angle of about 45° to the tracheid axis during formation of the secondary wall. Artificial inclination altered the pattern of alignment of MTs. Banding MTs were helically oriented late during the formation of the secondary walls. These results indicate that MTs might control the orientation and localized deposition of cellulose microfibrils in the secondary walls of compression wood tracheids.Part of this report was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 1996     

WATER CUTTING OF CHINESE ROSE-BETTY PRIOR

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Ultrastructure of the S2 layer in relation to lignin distribution inPinus radiata tracheids

The ultrastructure of the S₂ layer in relation to its lignin distribution was examined using transmission electron microscopy in the tracheids ofPinus radiata. The S₂ layer had a striated appearance at low magnification. Observations at higher magnifications showed lignin to be distributed inhomogeneously in this layer, appearing as a mosaic of electron-dense and electron-lucent regions. These regions are scattered, showing a pattern of often interconnecting sinuous features in a predominantly radial profile. The significance of these features of the S₂ layer is discussed, particularly in relation to the available information from recent ultrastructural observations on the appearance of cellulose microfibrils and the pattern of their distribution in the S₂ layer using rapid freeze-deep etching in conjunction with transmission electron microscopy. Predictions are made as to the likely distribution and arrangement of cellulose microfibrils in the S₂ layer based on the pattern of lignin distribution observed in this layer.     

Studies on compression wood—part VII: Distribution of lignin in normal and compression wood of tamarack [Larix laricina (Du Roi) K. Koch]

Summary The distribution of lignin has been studied in tracheids and ray cells of normal and compression wood of tamarack [Larix laricina (Du Roi) K. Koch]. The three layers in the secondary wall of normal wood tracheids are lignified to approximately the same extent, and previous evidence that the S ₃ layer should contain a higher proportion of lignin than the other regions has not been confirmed. The lignin follows closely the orientation of the cellulose microfibrils in all three layers. Compared to the tracheids, the ray cells contain a denser network of lignin in their secondary wall.Only a small proportion of the total lignin in compression wood tracheids is present in the compound middle lamella. The thick S ₁ layer is only slightly lignified; the orientation of the lignin in this region is that of the transversely oriented, lamellated microfibrils. The outer portion of S ₂ consists largely of lignin but also contains lamellae of cellulose microfibrils which probably have the same helical orientation as the microfibrils in the inner part of S ₂. The latter region, which contains the helical cavities, consists of lamellae of cellulose microfibrils which are uniformly encrusted with lignin. The ray cells in compression wood appear to be lignified to the same extent as in normal wood. Transverse sections of the cells reveal a lateral orientation of the lignin. The orientation of the cellulose microfibrils in the S ₂ layer of the first-formed springwood tracheids of compression wood is the same as in the cells which are formed later. It is suggested that for ease of reference, the outer, lignin-rich layer in compression wood tracheids be referred to as the S ₂(L) layer.

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Zusammenfassung Im Druckholz und im normalen Holz von Tamarack (Larix laricina (Du Roi) K. Koch) wurde die Verteilung des Lignins in Tracheiden und Markstrahlzellen untersucht. Die drei Schichten der Sekundärwand in den Tracheiden normalen Holzes werden in nahezu demselben Umfange lignifiziert. Frühere Feststellungen, daß die S ₃-Schicht einen höheren Ligningehalt erreicht als andere Zellwandbereiche, konnten also nicht bestätigt werden. Das Lignin folgt sehr genau der Orientierung der Cellulose-Mikrofibrillen aller drei Schichten. Im Vergleich zu den Tracheiden erfahren die Sekundärwände der Markstrahlzellen eine stärkere Ligninauskleidung.Nur ein geringer Prozentsatz des gesamten Lignins der Druckholztracheiden befindet sich in der Mittellamelle. Die dicke S ₁-Schicht ist nur wenig lignifiziert. Die Orientierung des Lignins in diesem Bereich entspricht den transversal orientierten, lamellierten Mikrofibrillen. Der äußere Teil der S ₂-Schicht enthält sehr viel Lignin, daneben aber auch Lamellen von Cellulose-Mikrofibrillen, die wahrscheinlich dieselbe spiralige Orientierung besitzen wie die Mikrofibrillen des inneren Teiles der S ₂-Schicht. Der letzterwähnte Bereich, der spiralige Kavitäten enthält, weist Lamellen von Cellulose-Mikrofibrillen auf, in welche gleichmäßig Lignin eingelagert ist. Die Markstrahlzellen des Druckholzes erscheinen ebenso stark lignifiziert wie die Markstrahlzellen des Normalholzes. Querschnitte durch diese Zellen lassen die laterale Orientierung des Lignins erkennen. Die Orientierung der Cellulose-Mikrofibrillen in der S ₂-Schicht der zuerst gebildeten Frühholztracheiden des Druckholzes ist dieselbe wie in jenen Zellen, die später ausgeformt werden. Es wird vorgeschlagen, daß zur eindeutigeren Kennzeichnung die äußere ligninreiche Schicht der Druckholztracheiden als S ₂(L)-Schicht bezeichnet wird.

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The authors wish to express their gratitude to Messrs. A. K. Bentum, D. C. Jones, and B. W. Simson for technical assistance. They are also thankful to Dr. D. A. I. Goring, McGill University, Montreal, Canada, for valuable discussions and for making available to them important, unpublished information. This investigation was supported by the United States Department of Agriculture, Forest Service, through Forest Service Research Grant No. 1, which is hereby gratefully acknowledged.     

The Delignification Pattern of Ailanthus excelsa Wood by Inonotus hispidus (Bull.: Fr.) P. Karst.

In vitro laboratory decay tests on Ailanthus excelsa Roxb. wood revealed that I. hispidus exhibits a combination of both white-rot and soft-rot patterns of wood decay. Early stages of wood decay showed dissolution of the middle lamella as well as defibration and localized delignification of fiber walls; vessels, axial, and ray parenchyma remained unaltered. Delignification commenced from the middle lamellae at the cell corners without any marked effect on the primary and secondary wall layers. In later stages of growth, the species produces typical soft-rot decay pattern by forming erosion channels through the S₂ layers of fiber walls, transverse bore holes in the cell walls, and erosion channels alongside/following the orientation of cellulose microfibrils. The rays showed signs of cell wall alterations only after the extensive damage to the fiber walls. After 120 days of incubation, the vessels also showed localized delignification, the erosion of pits, and separation from associated xylem elements. The extensive weight losses under natural and in vitro decayed wood as well as the very soft nature of severely degraded wood indicate that I. hispidus alters wood strength and stiffness.     

The supply of matrix containing glucomannans to the innermost surface of S2 layers is associated with changes in turgor pressure of differentiating tracheids in Cryptomeria japonica

We investigated the relationship between turgor pressure and diurnal differences in secondary wall formation of differentiating tracheids. Saplings of Cryptomeria japonica were grown in a growth chamber with 12-h light:12-h dark cycles, and the tangential strain on the inner bark surface was measured as an indicator of the volumetric changes of differentiating cells. The innermost surface of developing secondary walls was then observed using field emission scanning electron microscopy at 1-h intervals after both light and dark periods. Dramatic changes in the aspects of the innermost surface of developing secondary walls occurred 3h after the light was switched on and 4h after the light was switched off. The amorphous material containing glucomannans became evident when the differentiating cells became fully turgid during the dark period. Conversely, cellulose microfibrils became clearly visible when the cell volume was low during the light period. These results suggest that the diurnal periodicity in the supply of hemicellulose-containing matrix to developing secondary walls is associated with the changes in turgor pressure of differentiating tracheids that result from the change in light conditions during the photoperiodic cycle.     

Mechanical interaction between cellulose microfibril and matrix substance in wood cell wall determined by X-ray diffraction

We investigated mechanical interactions between the cellulose microfibril and the matrix substance in wood cell walls. X-ray diffraction measurements showed that the peak positions of (200) and (004) from cellulose crystals in wood cell walls tended to shift lower and higher toward 2θ, respectively, during water desorption in wood. From our simulations, it is shown that the peak shift of (200) during water desorption is not due to changes in the scattering pattern of the amorphous substance or to lateral expansion of the cellulose crystals due to the Poisson effect in the cellulose microfibril, which is compressed in the molecular chain direction as the amorphous substance shrinks. This suggests that the cellulose microfibril expands transversely during water desorption in the wood cell wall, and that there is a mechanical interaction between the cellulose microfibril and the matrix substance.     

The formation of LC and other aromatic end-group structures in <Emphasis Type="Italic">O</Emphasis>-alkyl-substituted cellulose during kraft pulping conditions

Ultraviolet resonance Raman spectroscopy (UVRRS) was used to study the formation of aromatic and/or lignin-carbohydrate (LC) structures at the reducing end groups of O-alkyl-substituted cellulose under conditions simulating the initial phase of kraft pulping. The derivatives studied were methyl cellulose (MC) with degree of substitution (DS) of 1.64–1.95, carboxymethyl cellulose (CMC) with DS ∼0.6, and a lignin model compound, creosol. The total alkali concentrations in the treatments were 0.1 M and 0.5 M and the sulfidities were 1%, 10%, and 30%. HS⁻ ions and creosol are both strong nucleophiles and they compete for the hot-alkali-generated unsaturated electrophilic reaction sites in the reducing end groups of the polysaccharides. The results indicated that conditions similar to those in the initial phase of conventional kraft cooking (high OH⁻ and low HS⁻ ion concentrations) increased the aromatic nature of the end groups and conditions similar to those in the initial phase of super batch cooking (low OH⁻ and high HS⁻ ion concentrations) partly inhibited the formation of aromatic and LC end groups.     

Infektion und Abbau des Wurzelholzes von Fichte durch Fornes annosus1

Infection and Deterioration of Spruce Root Wood by Fornes annosus . The paper describes the mode of infection and subsequent deterioration of spruce root wood by Fornes annosus using light and electronmicroscopic observations. In the secondary xylem the overall penetration of cell walls is effected by means of microhyphae which produce boreholes. The degradation of cell walls is initiated in the S₂ layer by diffusion of enzymes along the cellulose fibrils with subsequent hydrolysis of the amorphic incrusting substances.     

A histological investigation of Oriental beech wood decayed by Pleurotus ostreatus and Trametes versicolor

Chemical, light and electron microscopic studies were carried out on wood of Oriental beech (Fagus orientalis Lipsky) decayed by the white‐rot fungi Pleurotus ostreatus and Trametes versicolor for 30, 60 and 120 days according to the modified European standard EN 113. Mass loss as well as lignin, cellulose and carbohydrate content were determined before and after fungal attack. There were no significant differences of wood mass loss and chemical composition between both fungi at the end of incubation. After each incubation period, small specimens were stained for microscopic studies. The micromorphology of fungal cell wall degradation was rather similar for both fungi. Both decreased the cell wall thickness to the same extent. The accumulation of hyphae as well as the rupture of cell walls was also similar. The occurrence of hyphae, cavities in the pits and vessel walls followed nearly the same patterns. The parenchyma cells were completely destroyed. Altogether, both fungi produced a simultaneous white rot in Oriental beech wood.     

Small-angle X-ray scattering study on nanostructural changes with water content in red pine,American pine,and white ash

Wood is a highly sophisticated and multihierarchical material. The nanoscale structures in natural cell walls of red pine, American pine, and white ash specimens were investigated using the small-angle X-ray scattering (SAXS) technique. A tangent-by-tangent method was used to analyze the SAXS data. The results demonstrate that the multihierarchical scatterers in the three specimens can be divided into two dominant components, i.e., a sharp component and a wide component. The sharp component mainly corresponds to the contribution of cellulose microfibrils, and its size is almost unaffected by the water content. However, the wide component includes voids or microcracks and cellulose microfibril aggregates; its size changes, reflecting swelling and water accumulation in the voids or microcracks. Because of the different morphological features of the cell walls, softwood (red pine and American pine) displays different tendencies from hardwood (white ash) in terms of changes in the wide component with water content: the average scatterer size of the wide component has an incremental tendency with the water content in softwood, but it has a descending tendency in hardwood. Fractal analysis further revealed that in white ash the surface of scatterers is coarser and the scatterers form more compact nanostructures than in the two pine woods. All this nanostructural information can be used to explain well the difference of swelling behaviors between the two pines and the white ash.     

A contribution to the ultrastructure of tension wood fibers

Summary Pronounced tension wood from four North-American hardwood species has been examined by light and electron microscopy. Delignified fibers were also studied. The gelatinous layer was in all cases loosely attached to S₂ but varied considerably in thickness within each species and was in one case terminated towards the lumen by a layer resembling S₃. A terminal lamella was not observed. After considering both earlier evidence and the present results, it was concluded that the gelatinous layer has neither a honeycomb nor a homogeneous texture, as has been suggested, but that it consists of concentric lamellae of cellulose microfibrils. In the absence of hemicelluloses and lignin, the microfibrils are probably bound together less firmly than they are in other cell wall layers. The gelatinous layer is more readily separated from the remainder of the cell wall by mechanical forces than by chemical reagents.

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Zusammenfassung Zugholz von vier nordamerikanischen Laubbäumen wurde im Licht-und im Elektronenmikroskop ebenso wie entlignifizierte Fasern untersucht. Die gelatinöse Schicht war überall locker an die S₂ gebunden, zeigte aber sehr verschiedene Dicken und war in einem Fall gegen das Lumen von einer Schicht, die der S₃ ähnlich sah, begrenzt. Eine Abschlußlamelle konnte nicht beobachtet werden. Frühere und die eigenen Resultate zeigten, daß die gelatinöse Schicht weder eine Wabenstruktur noch eine homogene Struktur besitzt, sondern daß sie aus konzentrischen Lamellen von Cellulosemikrofibrillen besteht. Da Hemicellulosen und Lignin nicht vorhanden sind, sind die Mikrofibrillen wahrscheinlich nicht so fest aneinander gebunden wie in den anderen Zellwandschichten. Die gelatinöse Schicht läßt sich von der übrigen Zellwand durch mechanische Kräfte leichter als durch chemische Reagenzien trennen.

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This investigation was supported by the United States Department of Agriculture, Forest Service, through Forest Service Research Grant No. 1, which is hereby gratefully acknowledged.