Individual Cellulose Research Articles

Longitudinal and concurrent dimensional changes of cellulose aggregate fibrils during sorption stages

Atomic force microscopy (AFM) studies of the dimensional changes of cellulose microfibril materials, called cellulose aggregate fibrils (approx. 100 µm × 3 µm × 300 nm), exposed to two distinct relative humidities of 80% and 23% for 24 h and then suddenly subjected to 50% RH and 23 °C show that the fibrils are responsive to the surrounding environments in a nonspecific fashion. AFM images (10 µm × 10 µm) of the individual straight cellulose aggregate fibrils were taken as a function of elapsed time during both desorption and adsorption of moisture. The longitudinal distance between discrete natural defects observed on the cellulose aggregate fibrils as well as the width, cross-sectional area, and height of the cellulose aggregate fibril were measured from the AFM images. The length of the cellulose aggregate fibrils was found to have reduced after exposure to either high or low relative humidity, and then placement in ambient conditions. Over time in ambient conditions, the cellulose aggregate fibrils progressively relaxed to their original length during both desorption and adsorption of moisture. However, the relaxation rate during adsorption was faster than that during desorption. The possible explanations for this phenomenon are discussed including the sample preparation method, volume conservation, entropy elasticity, and free volume theory. The changes in the width, height, and cross-sectional area are also discussed.

Relationship of wood composition to growth traits of selected open-pollinated families of Eucalyptus urophylla from a progeny trial in Vietnam

Lignin and cellulose contents and wood basic density were related to diameter at breast height (DBH) in six fast-growing and five slow-growing families from a combined progeny test and seedling seed orchard of Eucalyptus urophylla grown for 10 years in northern Vietnam. The mean cellulose content of the fast-growing families was significantly higher than that of the slow growing-families (40.0 and 37.1%, respectively), and for individual trees cellulose content was significantly correlated phenotypically with DBH. Wood basic density was significantly lower in the fast-growing group than in the slow-growing group (0.506 and 0.535 g cm−3, respectively), and was significantly negatively correlated phenotypically with DBH. The lignin contents were not significantly different between groups. Cellulose content and wood basic density were not correlated. The main conclusion is that there is no obstacle to combining high growth rate with high cellulose content, for plantation of forests intended mainly for pulpwood.

Mechanism of cellulase reaction on pure cellulosic substrates

Cellulase reaction mechanism was investigated with the use of following pure cellulosic substrates: Microcrystalline cellulose (Avicel), alpha-cellulose (Sigma), filter paper, cotton, and non-crystalline cellulose (NCC). NCC is amorphous cellulose prepared in our laboratory by treatment with concentrated sulfuric acid. When hydrolyzed with cellulase, NCC produces significant amount of cello-oligosaccharides (COS) as reaction intermediates along with glucose and cellobiose. The COS produced by cellulase were categorized into two different moieties based upon their degree of polymerization (DP): low DP (less than 7) COS (LD-COS) and high DP COS (HD-COS). Endo-glucanase (Endo-G) reacts rapidly on the NCC reducing its DP to 30-60, after which the Endo-G reaction with NCC ceases. HD-COS is produced from NCC by the action of Endo-G, whereas LD-COS is produced by exo-glucanase (Exo-G). beta-Glucosidase (beta-G) hydrolyzes LD-COS to produce cellobiose, but it does not hydrolyze HD-COS. DP of NCC affects the action of Exo-G in such a way that the overall yield is high for high DP NCC. This is in line with previous findings that substrate-recognition by Exo-G requires binding on beta-glucan chain with DP of 10 for the hydrolysis to take place. The individual cellulose chain residues within solid having DP less than 10 therefore remain unreacted. The percentage of the unreacted portion would be lower for high DP NCC, which results high overall conversion. The surface area and the number of reactive sites on the substrate facilitate adsorption of enzyme therefore the initial rate of the hydrolysis. The overall extent of conversion of cellulose, however, is controlled primarily by its inherent characteristics such as DP and crystallinity.

Capturing the portrait of isolated individual natural cellulose molecules

Natural cellulose molecules have a strong tendency of being aggregated into larger structures. Thus, the imaging of isolated individual cellulose molecules is hampered for a long time. In this work, we manage to observe, for the first time, the isolated individual natural cellulose chains on a sample surface by means of atomic force microscope. The advantage of the ionic liquid, in which natural cellulose can be molecularly dispersed, is considered to be the key point for the successful imaging. Moreover, we find that the surface charge can influence the morphology of the single cellulose chains upon adsorption. That is, on the positively charged surface, individual cellulose chains adopt an extended conformation; whereas on the negatively charged surface, a compact globule conformation is observed.

Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Cellulases, enzymes capable of depolymerizing cellulose polymers into fermentable sugars, are essential components in the production of bioethanol from lignocellulosic materials. Given the importance of these enzymes to the evolving biofuel industry considerable research effort is focused on understanding the interaction between cellulases and cellulose fibrils. This manuscript presents a method that addresses challenges that must be overcome in order to study such interactions through high-resolution fluorescence microscopy. First, it is shown that cellulose can be immobilized on solid substrates through a polymer lift-off technique. The immobilized cellulose aggregates present characteristic morphologies influenced by the patterned feature size used to immobilize it. Thus, through a variety of pattern sizes, cellulose can be immobilized in the form of cellulose particles, cellulose mats or individual cellulose fibrils. Second, it is shown that both cellulose and Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A can be fluorescently tagged and that the labeling does not inhibit the capability of these cellulases to depolymerize cellulose. The combination of the immobilization technique together with fluorescence labeling yields a system that can be used to study cellulose-cellulase interactions with spatial and temporal resolution not available through more conventional techniques which measure ensemble averages. It is shown that with such a system, the kinetics of cellulase binding onto cellulose fibrils and mats can be followed through sequences of fluorescence images. The intensity from the images can then be used to reconstruct binding curves for the cellulases studied. It was found that the complexity of cellulose morphology has a large impact on the binding curve characteristics, with binding curves for individual cellulose fibrils closely following a binding saturation model and binding curves for cellulose mats and particles deviating from it. The behavior observed is interpreted as the effect pore and interstice penetration play in cellulase binding to the accessible surface of cellulose aggregates. These results validate our method for immobilizing nanoscale cellulose fibrils and fibril aggregates on solid supports and lay the foundation for future studies on cellulase-cellulose interactions.

Indirect evidence of supramolecular changes within cellulose microfibrils of chemical pulp fibers upon drying

Dried and never-dried chemical pulps were subjected to strong sulfuric acid hydrolysis and the dimensions of the resulting cellulose nanocrystals (CNCs) were characterized by AFM image analysis. Although the average length of CNCs was fairly similar in all samples (55–65 nm), the length distribution histograms revealed that a higher number of longer crystals and a lower number of shorter crystals were present in the CNC suspensions prepared from never-dried pulps. The distinction was hypothetically ascribed to tensions building in individual cellulose microfibrils upon drying, resulting in irreversible supramolecular changes in the amorphous regions. The amorphous regions shaped by tensions were deemed as more susceptible to acid hydrolysis.

Molecular imaging of single cellulose chains aligned on a highly oriented pyrolytic graphite surface

Individual cellulose macromolecules were successfully visualized on a highly oriented pyrolytic graphite (HOPG) surface by tapping-mode atomic force microscopy under ambient condition. Monomolecular-level dispersion of cellulose chains was achieved through the momentary contact of dilute cellulose/cupri-ethylenediamine (Cu-ED) solution onto the HOPG substrate. Both concentrations of cellulose and Cu-ED provided critical impacts on the topographical images. Single cellulose chains with molecular height of ca. 0.55 nm could be observed under the optimal conditions, showing rigid molecular rods with a unique morphology of hexagonal regularity. It was strongly suggested that the cellulose chains were aligned along the HOPG crystal lattice through a specific attraction, possibly due to a CH–π interaction between the axial plane of cellulose and the HOPG π-conjugated system. These phenomena would imply the potential applications of an HOPG substrate for not only nano-level imaging, but also for molecular alignment of cellulose and other structural polysaccharides.

Dual-axis electron tomography: a new approach for investigating the spatial organization of wood cellulose microfibrils

We have employed dual-axis electron tomography to investigate the 3D organization of cellulose microfibrils in plastic resin-embedded, delignified cell walls of radiata pine early wood. The ∼ 1 nm thick tomographic slices produced in this study provided for a resolution of ∼ 2 nm in the cross-section of the slices throughout the 150 nm thick plastic sections. This resolution is sufficient to resolve individual cellulose microfibrils and to map the 3D organization of the cellulose microfibrils within the S2 layer of the secondary cell walls. The individual cellulose microfibrils measure ∼ 3.2 nm in diameter, and appear to consist of a ∼ 2.2 nm unstained core and a ∼ 0.5 nm thick surface layer that is lightly stained. Both individual and clustered cellulose microfibrils are seen surrounded by more heavily stained and irregularly shaped residual lignin and hemicellulose. The tightness of packing of the cellulose microfibrils in the cluster varies along the thickness of the section. These findings demonstrate that dual-axis electron tomography is a technique that can provide new insights into the 3D organization of cellulose microfibrils in plant cell walls.

Conducting polymer composites with cellulose and protein fibres

Conducting polymer–fibre composites with polypyrrole cellulose (paper) and protein (wool) fibres have been produced by polymerizing pyrrole with ferric chloride as the oxidant around separated individual cellulose (wood) fibres from bleached Pinus radiata Kraft pulp, and merino wool fibres respectively to generate new composite materials that retain the inherent properties of the polymer and individual fibres. For the polypyrrole–cellulose composite, the polypyrrole consisted of spheres of about 100 nm in size fused together to form a continuous coating that fully encapsulates the cellulose fibre and associated finer fibrils. For the polypyrrole–wool composite the polypyrrole formed a similar coating of about 200 nm spheres. Electrical conductivities of the composites were substantially increased over those of the precursor fibres.

Influence of xylan on the degradability of laboratory kraft pulps from hardwood and reed canary grass in acid hydrolysis

The depolymerisation of laboratory-prepared kraft pulps from birch, eucalyptus and reed canary grass during acid hydrolysis was studied. The intention was to study especially the influence of xylan content on the levelling-off degree of polymerisation (LODP) and on the dissolution of carbohydrates during the acid hydrolysis. The xylan content in the pulps was varied by prehydrolysis prior to the kraft pulping or by alkali-extraction of the bleached pulps, and the levelling-off degree of polymerisation was compared with the amount of xylan left in the pulps at LODP. The dimensions of the original fibers in the pulps and of the fiber fragments after hydrolysis were also measured. It was found that the fiber fragments after hydrolysis were longer in the pulps containing a higher amount of xylan. Xylan thus appears to prevent degradation during acid hydrolysis, both on the fiber level and on the individual cellulose fibril level.

Conducting Polymers on Paper Fibres

Conducting polymers have generated a great deal of interest because of their physical and chemical properties as well as their potential in industrially useful materials. However, one of the shortcomings of most conducting polymers is that they are often formed as intractable films that are difficult to process [1]. To overcome this problem we have incorporated various conducting polymers into sheets of paper in order to create new composite materials which combine the universal properties of a paper sheet or paper products with the chemical and electrically conducting properties of the conducting polymer. Various composite paper – conducting polymer products have been prepared by polymerizing polypyrrole and polyaniline directly onto the paper sheet using ferric chloride as the oxidant. Electronmicroscopy shows that the polymers comprise spheres of about 50–150 nanometers in size fused together such that they fully encapsulate the individual cellulose fibres in the paper sheet. The open matrix of fibres characteristic of the original paper sheet is maintained and hence provides a much greater specific surface area for the conducting polymer than that of a continuous flat sheet which would be the case if the polymer simply formed a laminar coating on the paper surface. In the case of polyaniline, it was also possible to form a different morphology of the polymer comprising a mix of nano-size spheres, rods and plates using ammonium persulfate as the oxidant. Conductivities of up to 6 S cm −1 have been measured for paper-polypyrrole composites and up to 2x10 −3 S cm −1 for paper-polyaniline composites.

Direct Force Measurements between Cellulose Surfaces and Colloidal Silica Particles

The interaction of cellulose layers with colloidal silica particles was investigated by direct force measurements with the atomic force microscope (AFM). Upon approach, repulsive forces were found between the negatively charged silica particles and the cellulose surface. The forces were interpreted quantitatively in terms of electrostatic interactions due to overlap of diffuse layers originating from negatively charged carboxylic groups on the cellulose surface. The diffuse layer charge density of cellulose was estimated to be 0.80 mC/m2 at pH 9.5 and 0.21 mC/m2 at pH 4. The forces upon retraction are characterized by molecular adhesion events, whereby individual cellulose chains desorb from the probe surface. The retraction profiles are dominated by well-defined force plateaus, which correspond to single-chain desorption forces of 35-42 pN. We surmise that adsorption of cellulose to probe surfaces is dominated by nonelectrostatic forces, probably originating from hydrogen bonding. Electrostatic contributions to desorption force could be detected only at high pH, where the silica surface is highly charged.

Measurement of the Elastic Modulus of Single Bacterial Cellulose Fibers Using Atomic Force Microscopy

The ability of the atomic force microscope to measure forces with subnanonewton sensitivity at nanometer-scale lateral resolutions has led to its use in the mechanical characterization of nanomaterials. Recent studies have shown that the atomic force microscope can be used to measure the elastic moduli of suspended fibers by performing a nanoscale three-point bending test, in which the center of the fiber is deflected by a known force. We extend this technique by modeling the deflection measured at several points along a suspended fiber, allowing us to obtain more accurate data, as well as to justify the mechanical model used. As a demonstration, we have measured a value of 78 +/- 17 GPa for Young's modulus of bacterial cellulose fibers with diameters ranging from 35 to 90 nm. This value is considerably higher than previous estimates, obtained by less direct means, of the mechanical strength of individual cellulose fibers.

Nanocoating of natural cellulose fibers with conjugated polymer: hierarchical polypyrrole composite materials

Astonishingly uniform polypyrrole nanocoating on morphologically complex cellulosic substances was achieved without disrupting the hierarchical network structures of individual cellulose fibers by means of polymerization-induced adsorption.

Effect of Two Systemic Fungicides on Cellulose Decomposing Fungi of Tomato Plants and on Some Enzymatic Activities

Kocide 101 (77% cupric hydroxide) and Ridomil plus (15% metalaxyl and 35% copper oxychloride) were used in the treatment of tomato plants. The two fungicides exerted a depressive effect on the total counts and on the individual cellulose decomposing fungal species associated with the roots and shoots of tomato. When these fungicides were incorporated in the liquid culture medium specified for growth and extracellular enzyme production by some selected fungal species, there was a significant reduction in mycelial growth as well as in amylase, cellulase, lipase and protease production by the fungi tested, particularly at the higher doses (200-400 ppm). Exceptions were observed with lower doses (50 and 100 ppm) especially in case of Aspergillus flavus, Cunninghamella echinulata, Penicillium chrysogenum and Fusarium oxysporum grown for amylase or cellulase production.

Evaluation of elastic modulus of materials by adhesive (no–slip) nano–indentation

Results of Mossakovskii's analysis for the elastic, adhesive (no–slip) contact between a rigid, axisymmetric punch and an isotropic, elastic half–space are developed in order to show that a simple relation exists between the contact stiffness, the contact area and the elastic modulus. The relation is similar to the Bulychev–Alekhin–Shorshorov equation, which is commonly used for evaluation of elastic modulus of materials by nano–indentation. The final formula differs from the frictionless case by a factor that depends on the Poisson ratio of the material. The bounds for the values of the factor are estimated. Similar to the frictionless analysis performed by Pharr, Oliver and Brotzen, the relation is not dependent on the geometry of the punch.

Characterisation of the microstructure and deformation of high modulus cellulose fibres

This paper reports on the microstructure and deformation of one type of high modulus cellulose fibre characterised using the techniques of Raman spectroscopy and synchrotron X-ray diffraction and it compares this fibre to a lower modulus conventional viscose fibre. The crystallinity of the fibres has been determined using X-ray diffraction. The orientation parameter has been determined by measuring the width of the (200) equatorial reflections for each fibre using microfocus synchrotron radiation and it has also been shown that the crystal orientation parameter varies from the skin to core of the fibres and is different for each type. Mechanical properties of the fibres are reported and it is shown that the high modulus cellulose fibres have very different stress–strain behaviour to the viscose fibres. Finally, it is shown for the high modulus fibre that the 1414, 1260, 1095 and 895 cm−1 Raman bands shift under the application of tensile deformation towards a lower wavenumber with the 1095 cm−1 band giving information about the backbone chain stretching of the cellulose. The viscose fibres show less significant shifts in this peak. The crystal modulus of the high modulus cellulose fibre has also been determined by calculating the change in the c-spacing upon the application of tensile deformation to individual cellulose monofilaments. This change in the c-spacing is determined from the change in position of the (002) meridional reflection giving a crystal modulus of 77 GPa. This value is a little low compared to other published data, and reasons for this are discussed. The shear modulus between crystallites is also calculated and compared to previously published data.

Molecular Dynamics Simulations of Bulk Native Crystalline and Amorphous Structures of Cellulose

Molecular modeling has been performed on three cellulosic systems: the two native crystalline phases (Iα and Iβ) and an amorphous phase, constituted by four independent microstructures. The goal of the study is to describe different organizations of the material and to emphasize how crystalline and amorphous celluloses differ. Besides, the study of the crystal structures for which many experimental data are available allows an estimation of the ability of the force field to model condensed phases of cellulose. For these organized structures the bulk parameters, such as unit cell dimensions, densities, Hildebrand solubility parameters, and hydrogen bonding, compare favorably well with the available experimental measures. The individual cellulose chains conformational behavior is as expected: torsion angles of the glycosidic bonds explore the g-values, hydroxymethyl groups are in the tg orientation, and the pyranoid ring puckering is in the 4C1 chair form. On the contrary, all the conformational parameters of the amorphous models show large variations: preferred values of the glycosidic bond torsion angles reproduce, however, the potential energy surface of cellobiose model compound. Furthermore, the Φ torsion angle behavior is in good concordance with the exo-anomeric effect. Hydroxymethyl groups explore mostly the gg and gt orientations, and high-energy puckering of the pyran rings is stabilized within the amorphous solid. Interchain interactions on both amorphous and crystalline structures are analyzed by means of the hydrogen bonding network. Finally, estimation of the glass transition temperature of an amorphous microstructure is given.

Micromechanical characterization of cellulose fibers

The work presented here aims towards the characterization of micromechanical properties of individual cellulose fibers. In particular, our interest is in determining the elastic modulus, tensile strength and tensile elongation of cotton and wood based cellulose fibers. Testing is conducted at a microscale, on single fibers of a few millimeters in length and tens of microns in width, a scale so chosen because such fibers are commonly used as industrial raw materials. An in-house microtensile fiber tester was designed and fabricated. A fine resolution stepper motor (10 nm per step) provides the actuation for the microtensile load frame and load in the fibers is measured by a high sensitivity load cell (0.01 mN resolution). The entire assembly is placed below a zoom stereo microscope for the dual purpose of aiding in specimen mounting and visually recording the experiment. After calibration of the device, good results were obtained when testing both the cotton and wood based cellulose fibers. Average Young's moduli of 4 GPa and 2 GPa were found for the two fibers, respectively. However, a large variation in failure stress was found for the fibers of both kinds, necessitating a statistical analysis assuming a Weibull distribution of failure. Based on this, mean strengths of 496 MPa for cotton cellulose fibers and 164 MPa for wood cellulose fibers were obtained.

In vitro and in vivo evaluation of e‐PTFE and alkali‐cellulose membranes for guided bone regeneration

Guided bone regeneration (GBR) is employed to encourage the formation of new bone in osseous defects by restricting the infiltration of soft tissues. While a variety of membranes have been evaluated for this surgical procedure, the non-resorbable material of choice is currently expanded polytetrafluoroethylene (e-PTFE). A new alkali-cellulose membrane produced by a biotechnological process has been developed as an alternative to e-PTFE for GBR. In this study, the biocompatibility of this novel alkali-cellulose membrane and e-PTFE was compared using tissue culture and an in vivo GBR model. In vitro both materials supported the attachment, migration and differentiation of osteoblast-like cells in culture for up to 3 weeks. The in vivo model was based upon full-thickness transcortical bone defects in the mandibular rami of Sprague-Dawley rats. The right rami were used as controls, contralateral defects being covered bucally and lingually with either e-PTFE or alkali-cellulose membranes. Pathological and histomorphometric analysis was undertaken at 4 and 10 weeks post-implantation. Bone regeneration associated with alkali-cellulose membranes was predominantly endochondral in type in contrast to e-PTFE which induced direct bone formation (intramembranous ossification). The amount of new bone formed in defects was similar for both types of membrane, but alkali-cellulose membranes induced significantly greater inflammatory response; characterized by lymphocytes, macrophages and multinucleated giant cells. Degradation and possible exposure of individual cellulose fibres may account for the poor performance of alkali-cellulose membranes in vivo. This animal and in vitro study indicates that when choosing a non-resorbable membrane for GBR, e-PTFE membranes are likely to perform better than those produced from alkali-cellulose.