Cellulose Microfibril Bundles Research Articles

An Oxidative Enzyme Boosting Mechanical and Optical Performance of Densified Wood Films (Small 17/2023)

Densified Wood Films In article number 2205056, Qi Zhou and co-workers show that cellulose-oxidizing enzyme lytic polysaccharide monooxygenase (LPMO) can nanofibrillate cellulose microfibril bundles inside the wood cell wall, altering wood microstructure. Therefore, delignified wood can be densified under ambient temperature and low pressure into transparent, foldable, and ultrastrong films. This enzymatic approach offers a sustainable alternative for top-down modification of wood materials.

DEFECTIVE KERNEL1 regulates cellulose synthesis and affects primary cell wall mechanics.

The cell wall is one of the defining features of plants, controlling cell shape, regulating growth dynamics and hydraulic conductivity, as well as mediating plants interactions with both the external and internal environments. Here we report that a putative mechanosensitive Cys-protease DEFECTIVE KERNEL1 (DEK1) influences the mechanical properties of primary cell walls and regulation of cellulose synthesis. Our results indicate that DEK1 is an important regulator of cellulose synthesis in epidermal tissue of Arabidopsis thaliana cotyledons during early post-embryonic development. DEK1 is involved in regulation of cellulose synthase complexes (CSCs) by modifying their biosynthetic properties, possibly through interactions with various cellulose synthase regulatory proteins. Mechanical properties of the primary cell wall are altered in DEK1 modulated lines with DEK1 affecting both cell wall stiffness and the thickness of the cellulose microfibril bundles in epidermal cell walls of cotyledons.

An Oxidative Enzyme Boosting Mechanical and Optical Performance of Densified Wood Films.

Nature has evolved elegant ways to alter the wood cell wall structure through carbohydrate-active enzymes, offering environmentally friendly solutions to tailor the microstructure of wood for high-performance materials. In this work, the cell wall structure of delignified wood is modified under mild reaction conditions using an oxidative enzyme, lytic polysaccharide monooxygenase (LPMO). LPMO oxidation results in nanofibrillation of cellulose microfibril bundles inside the wood cell wall, allowing densification of delignified wood under ambient conditions and low pressure into transparentanisotropic films. The enzymatic nanofibrillation facilitates microfibril fusion and enhances the adhesion between the adjacent wood fiber cells during densification process, thereby significantly improving the mechanical performance of the films in both longitudinal and transverse directions. These results improve the understanding of LPMO-induced microstructural changes in wood and offer an environmentally friendly alternative for harsh chemical treatments and energy-intensive densification processes thusrepresenting a significant advance in sustainable production of high-performance wood-derived materials.

Nanoscale Mechanism of Moisture-Induced Swelling in Wood Microfibril Bundles.

Understanding nanoscale moisture interactions is fundamental to most applications of wood, including cellulosic nanomaterials with tailored properties. By combining X-ray scattering experiments with molecular simulations and taking advantage of computed scattering, we studied the moisture-induced changes in cellulose microfibril bundles of softwood secondary cell walls. Our models reproduced the most important experimentally observed changes in diffraction peak locations and widths and gave new insights into their interpretation. We found that changes in the packing of microfibrils dominate at moisture contents above 10–15%, whereas deformations in cellulose crystallites take place closer to the dry state. Fibrillar aggregation is a significant source of drying-related changes in the interior of the microfibrils. Our results corroborate the fundamental role of nanoscale phenomena in the swelling behavior and properties of wood-based materials and promote their utilization in nanomaterials development. Simulation-assisted scattering analysis proved an efficient tool for advancing the nanoscale characterization of cellulosic materials.

A TEMPO-catalyzed oxidation–reduction method to probe surface and anhydrous crystalline-core domains of cellulose microfibril bundles

A modified TEMPO-catalyzed oxidation of the solvent-exposed glucosyl units of cellulose to uronic acids, followed by carboxyl reduction with NaBD4 to 6-deutero- and 6,6-dideuteroglucosyl units, provided a robust method for determining relative proportions of disordered amorphous, ordered surface chains, and anhydrous core-crystalline residues of cellulose microfibrils inaccessible to TEMPO. Both glucosyl residues of cellobiose units, digested from amorphous chains of cellulose with a combination of cellulase and cellobiohydrolase, were deuterated, whereas those from anhydrous chains were undeuterated. By contrast, solvent-exposed and anhydrous residues alternate in surface chains, so only one of the two residues of cellobiosyl units was labeled. Although current estimates indicate that each cellulose microfibril comprises only 18 to 24 (1 → 4)-β-d-glucan chains, we show here that microfibrils of walls of Arabidopsis leaves and maize coleoptiles, and those of secondary wall cellulose of cotton fibers and poplar wood, bundle into much larger macrofibrils, with 67 to 86% of the glucan chains in the anhydrous domain. These results indicate extensive bundling of microfibrils into macrofibrils occurs during both primary and secondary wall formation. We discuss how, beyond lignin, the degree of bundling into macrofibrils contributes an additional recalcitrance factor to lignocellulosic biomass for enzymatic or chemical catalytic conversion to biofuel substrates.

Bundling of cellulose microfibrils in native and polyethylene glycol-containing wood cell walls revealed by small-angle neutron scattering

Wood and other plant-based resources provide abundant, renewable raw materials for a variety of applications. Nevertheless, their utilization would greatly benefit from more efficient and accurate methods to characterize the detailed nanoscale architecture of plant cell walls. Non-invasive techniques such as neutron and X-ray scattering hold a promise for elucidating the hierarchical cell wall structure and any changes in its morphology, but their use is hindered by challenges in interpreting the experimental data. We used small-angle neutron scattering in combination with contrast variation by poly(ethylene glycol) (PEG) to identify the scattering contribution from cellulose microfibril bundles in native wood cell walls. Using this method, mean diameters for the microfibril bundles from 12 to 19 nm were determined, without the necessity of cutting, drying or freezing the cell wall. The packing distance of the individual microfibrils inside the bundles can be obtained from the same data. This finding opens up possibilities for further utilization of small-angle scattering in characterizing the plant cell wall nanostructure and its response to chemical, physical and biological modifications or even in situ treatments. Moreover, our results give new insights into the interaction between PEG and the wood nanostructure, which may be helpful for preservation of archaeological woods.

Variation in Hemicellulose Structure and Assembly in the Cell Wall Associated with the Transition from Earlywood to Latewood in Cryptomeria japonica

The size of cellulose microfibril (CMF) bundles varies to interact with glucomannan/galactoglucomannan (GM/GGM). Arabino-4-O-methylglucuronoxylan (AGX) bonded CMF bundles coated with GM/GGM also have important roles in elaborating the distance between these components. Since the precise roles of GM/GGM and AGX are not clear, the elution analysis to evaluate the strength of the interaction between the cell wall were tried. Earlywood (EW) and latewood (LW) were separated in a Japanese cedar. The chemical components of cellulose, hemicellulose including GM/GGM and AGX, and lignin were almost the same in EW and LW. Slight differences in GM/GGM, the side-chain substitution in AGX and the ionic bond characteristics of glucuronic acid side chains were observed. Based on measurements of GM/GGM and AGX adhering to CMFs, there were more hemicelluloses forming strong hydrogen bonds in LW than in EW. The results showed that the highly assembled hemicellulose in LW produced a strong cell wall framework.

In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens

Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

Dilute acid pretreatment differentially affects the compositional and architectural features of Pinus bungeana Zucc. compression and opposite wood tracheid walls

Compression wood represents a unique challenge for biochemical processing of sustainable biomass to produce biofuels and bioproducts on account of its highly lignified tracheids relative to opposite wood. In the current work, differentiating response of Pinus bungeana Zucc. compression wood and opposite wood tracheid walls to dilute acid pretreatment was elucidated by combining chemical and microscopic approaches. Dilute acid pretreatment released greater amount of hemicelluloses from opposite wood (91.0%) than from compression wood (31.2%). The dissolution of hemicelluloses was not uniform and varied across the tracheid wall. In addition, for both compression wood and opposite wood the heterogeneity in lignin distribution was enhanced, probably attributed to the lignin migration and relocalization. The ultrastructural arrangement of the cell wall was also altered with the pretreated opposite wood tracheid wall exposing more apparent cellulose microfibril bundles and less abundant globular structures compared to compression wood. The dilute acid pretreatment demonstrated that the greater recalcitrance of compression wood probably originated from the higher lignin content and the resulting more compact structure of the cell wall.

Antibacterial hybrid materials fabricated by nanocoating of microfibril bundles of cellulose substance with titania/chitosan/silver-nanoparticle composite films.

Uniform ultrathin titania/chitosan composite films were coated on the cellulose microfibril bundles of natural cellulose substance (common commercial filter paper) by a layer-by-layer self-assembly process. Relying on the strong chelating ability of chitosan for metal ions, silver ions were abundantly adsorbed on the titania/chitosan composite film coated cellulose substance and were thereafter in situ reduced to silver nanoparticles (Ag-NPs) under UV irradiation. As such, hybrid cellulose/titania/chitosan/Ag-NP composite materials were obtained, which feature the same hierarchically fibrous structure as the initial cellulose substance. Meanwhile, the hybrid fibres in the composite materials exhibit a cable-like core-shell structure, displaying a cellulose microfibril bundle core as well as a nanometer-thick titania/chitosan/Ag-NP composite film shell with Ag-NPs (4-20 nm in diameter) well distributed. The antibacterial activities of the titania/chitosan/Ag-NP composite film coated cellulose materials were evaluated against both Gram-positive and Gram-negative bacteria, and the materials as such disinfected almost all the inoculated bacteria due to the intrinsic biocidal effect of titania composition, positively charged chitosan component and high loading content of Ag-NPs with small sizes, showing excellent antibacterial activities.

Ultrastructure of the innermost surface of differentiating normal and compression wood tracheids as revealed by field emission scanning electron microscopy

The ultrastructure of the innermost surface of Cryptomeria japonica differentiating normal wood (NW) and compression wood (CW) was comparatively investigated by field emission electron microscopy (FE-SEM) combined with enzymatic degradation of hemicelluloses. Cellulose microfibril (CMF) bundles were readily observed in NW tracheids in the early stage of secondary cell wall formation, but not in CW tracheids because of the heavy accumulation of amorphous materials composed mainly of galactans and lignin. This result suggests that the ultrastructural deposition of cell wall components in the tracheid cell wall differ between NW and CW from the early stage of secondary cell wall formation. Delignified NW and CW tracheids showed similar structural changes during differentiating stages after xylanase or β-mannanase treatment, whereas they exhibited clear differences in ultrastructure in mature stages. Although thin CMF bundles were exposed in both delignified mature NW and CW tracheids by xylanase treatment, ultrastructural changes following β-mannanase treatment were only observed in CW tracheids. CW tracheids also showed different degradation patterns between xylanase and β-mannanase. CMF bundles showed a smooth surface in delignified mature CW tracheids treated with xylanase, whereas they had an uneven surface in delignified mature CW tracheids treated with β-mannanase, indicating that the uneven surface of CMF bundles was related to xylans. The present results suggest that ultrastructural deposition and organization of lignin and hemicelluloses in CW tracheids may differ from those of NW tracheids.

Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process

Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5wt.% NaOH solution produced a twofold increase in Young’s modulus of processed BC sheet over untreated BC sheet. Further enhancements are achieved after a second treatment with 2.5wt.% NaOCl (bleaching). These treatments were carefully designed in order to prevent any polymorphic crystal transformation from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morphological and thermal stability of the processed films are also improved.

New Approaches to Cellulose Microfibril Isolation from Musaceae Agro-Industrial Residues

In this work, Musaceae isolated vascular bundles from rachis agro-industrial residues were evaluated as a potential source of cellulose microfibrils. For vascular bundle isolation, a biological retting was used. For cellulose microfibril isolation, two different alkaline treatments (sodium hydroxide or potassium hydroxide combined with bleaching and acid steps) were used in conjunction with a mechanical process. Cellulose microfibrils using both alkaline processes were successfully isolated, and the presence of non-cellulosic components, especially lignin and some hemicellulose as arabinose and galactose were reduced. In spite of an important amount of oxides being removed during the biological retting, XRF analysis revealed that calcium minerals were still present in the vascular bundles, and they can affect the cellulose microfibril isolation. AFM micrographs of isolated samples revealed cellulose microfibril bundles, and their presence can be associated with non-cellulosic components still present in the samples. Thermal analysis showed that when potassium hydroxide was used, a higher reduction of lignin was observed. Nevertheless, X-ray diffractions indicated no change in the crystallization pattern of cellulose I had occurred due to the isolation process used.

Preparation and mechanical properties of bacterial cellulose nanocomposites loaded with silica nanoparticles

Bacterial cellulose (BC), which is produced by Gluconacetobacter xylinus (Ga. xylinus) in culture, is made up of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. In the current studies, we used two processes to prepare nanocomposites of BC filled with silica particles. In Process I, Ga. xylinus was incubated in medium containing silica sol Snowtex 0 (ST 0, pH 2–4) or Snowtex 20 (ST 20, pH 9.5–10.0). The elastic modulus at 20 °C was improved by keeping the amount of silica in the nanocomposites below 4% when ST 20 was used and below 8.7% when ST 0 was used. This process allowed incorporation of 50% silica in BC. Inclusion of higher amounts of silica reduced the modulus at 20 °C and the strength of the nanocomposites below that of BC. X-ray diffraction measurements revealed that the silica particles disturb the formation of ribbon-shaped fibrils and affect the preferential orientation of the ( $$ 1\overline{1} 0 $$ ) plane. We also produced BC-silica nanocomposites by Process II, wherein the BC hydrogel was immersed in different concentrations of silica sols, allowing silica particles to diffuse into the BC hydrogel and lodge in the spaces between the ribbon-shaped fibrils. This method increased the modulus at 20°C and the strength compared to the BC matrix, but it was difficult to load the BC with more than 10% silica in this way.

Research note: Deposition patterns of cellulose microfibrils in flange wall ingrowths of transfer cells indicate clear parallels with those of secondary wall thickenings.

The arrangement of cellulose microfibrils and cortical microtubules in transfer cells depositing flange wall ingrowths have been determined with field emission scanning electron microscopy and immunofluorescence confocal microscopy. In xylem transfer cells of wheat (Triticum aestivum) stem nodes and transfer cells of corn (Zea mays) endosperm tissue, cellulose microfibrils were aligned in parallel bundles to form the linear wall ingrowths characteristic of flange ingrowth morphology. In both cell types, linear bundles of cellulose microfibrils were deposited over an underlying wall composed of randomly arranged microfibrils. Acid extraction of wheat xylem transfer cells established that flange ingrowths were composed of crystalline cellulose. Immunofluorescence labelling of microtubules in wheat xylem transfer cells showed that bundles of microtubules were positioned directly below and parallel with developing flange ingrowths, whereas more mature ingrowths were flanked by bundles of microtubules. These results show that the parallel organisation of cellulose microfibrils in flange wall ingrowths is similar to those in secondary wall thickenings in xylem elements, and that deposition of these structures in transfer cells is also likely to involve bundling of parallel arrays of microtubules. Our observations are discussed in terms of the possible role of microtubules in building flange-type wall ingrowths and the consequences in terms of predicted mechanisms required to build the fundamentally different reticulate-type wall ingrowths.

Cellulose synthesized by Acetobacter xylinum in the presence of plant cell wall polysaccharides

Acetobacter xylinum was cultured in Schramm–Hestrin (SH) medium containing glucuronoxylan (xylan medium) or pectin (pectin medium). Loose bundles of cellulose microfibrils were formed in the xylan medium. In contrast the cellulose ribbons formed in the pectin medium were the same as those normally formed in SH medium.The periodic acidthiocarbohydrazidesilver proteinate method indicated that positive reacted substances located along cellulose microfibrils formed in both mediums. Freeze-fracture and deepetching electron microscopy also revealed that polysaccharides exist around cellulose microfibrils. X-ray diffractometry and Fourier Transform In-frared spectroscopy demonstrated that the addition of xylan induced a change in the ratio of cellulose Iα and Iβ. Electron diffraction analysis revealed that xylan discontinuously affected the crystalline structure of cellulose microfibrils. Pectin did not have this effect. Glucuronoxylan in the medium prevented the assembly of cellulose

Suspensions of cellulose microfibrils from sugar beet pulp

The chemical composition of sugar beet pulp (SBP) was analyzed. In addition to some mineral, this product consisted essentially of polysaccharides with about one third cellulose, one third hemicellulose and one third pectin. After a mild alkaline disencrusting and bleaching treatments, most of the hemicelluloses and pectins could be removed to yield a cellulosic residue that presented itself as a dispersion of thin parenchymal cell-ghosts having ovoid or elongated shapes and dimensions ranging from 50 to 200 μm. The origin of these elements was traced back to the anatomy of the sugar beet root which was also investigated by scanning electron microscopy after critical point drying. At the ultrastructural level, the disencrusted SBP cell-ghosts consisted of a loose network of cellulose microfibrils, typical of parenchymal cell cellulose (PCC). This network could be disrupted by homogenization with a Manton–Gaulin apparatus to yield aqueous suspensions of either individual or bundles of cellulose microfibrils. This cellulose was characterized in terms of crystallinity, average degree of polymerization and chemical composition. The PCC suspensions did not flocculate nor sediment as long as some hemicellulose and pectin was maintained at the microfibril surface. Flocculation, however, occurred if these encrustants were removed by either strong alkali or a trifluoroacetic acid (TFA) treatment.

Sinuous ordinary epidermal cells: behind several patterns of waviness, a common morphogenetic mechanism.

Morphogenesis of sinuous epidermal cells in leaves of the fern Asplenium nidus and the monocotyledonous Cyperus papyrus, petals of the dicotyledonous Begonia lucerna. and in-vitro-grown leaves of the fern Adantum capillus-veneris is controlled by the local differentiation of their walls. In all these cases wall pads, including radial cellulose microfibrils, arc deposited at the junctions of the external periclinal wall with the anticlinal ones. Moreover, in Asplenium nidus, similar wall pads form at the junctions of the internal periclinal wall with the anticlinal ones. The wall pads are connected to anticlinal cellulose microfibril bundles running the whole depth of the anticlinal walls nr part of it. This wall differentiation imposes a highly controlled cell wall expansion, a consequence of which is the waviness of the epidermal cell anticlinal, walls. The pattern of wall reinforcement varies among different species, resulting in differences in the pattern of waviness. Cortical microtubule arrays mirror the orientated deposition of cellulose microfibrils in the epidermal Cells. These findings, derived from plants from different major groups, show a common epidermal cell morphogenetic mechanism depending on radial cellulose microfibrils and cellulose microfibril bundles. The facts that (a) epidermal cell morphogenesis in Adiantum copillus-veneris leaves grown in vitro differs considerably from that of typical leaves and (b) petal epidermal cells in Begonia lucerna are sinuous, while leaf epidermal cells are not, suggest that this mechanism may he affected by epigenetic factors.

Incipient decay of Quercus serrata sapwood by Lentinus edodes and its inhibition by an antagonistic hyphomycete, Leptodontidium elatius

Incipient decay of Quercus serrata by Lentinus edodes and its inhibition by Leptodontidium elatius were investigated by scanning electron microscopy. Rapid accumulation of L. edodes hyphae in earlywood vessels was a prerequisite to efficient decay of sapwood. In the early stages of decay, selective removal of lignin and other amorphous wall components occurred in the vessels and surrounding tissues, including vasicentric tracheids and wood fibres. Thus, bundles of cellulose microfibrils, having lateral dimensions of 15–50 nm, became recognizable. Disintegration of cellulose microfibril bundles appeared to begin with longitudinal and (or) transverse splitting of the bundles, and then the walls became progressively thinner. In ray parenchyma cells, end walls were susceptible to the fungal degradation, but lateral walls, particularly the innermost layer, were relatively resistant. Hyphae of L. elatius grew in close contact with the vessel wall and exuded slime material to create a distinct microarea (mycosphere) around them. In the vessels precolonized by this hyphomycete, the growth rate of L. edodes hyphae was drastically reduced to less than 20% of that in antagonist-free vessels. Possible roles of the slime material in this antagonism are discussed. Key words: wood decay, Lentinus edodes, Leptodontidium elatius, antagonism, mycosphere.

Orientation of cellulose microfibrils in cortical cells of tobacco explants

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations - all given with respect to the longitudinal axis of the cell -, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.