Helga C. Lichtenegger

Experimental evidence for a mechanical function of the cellulose microfibril angle in wood cell walls

Abstract Wood is a natural fibre composite with a hierarchical cellular structure of a specific strength and a specific modulus of elasticity that can be compared with those of other common construction materials. Each wood cell is typically built of cellulose fibrils spiralling around the macroscopic fibre direction. While it is natural to assume a relation between the microfibril angle (MFA) and the mechanical properties, a good correlation has up to now only been established for single fibres, where a larger extensibility was found for fibres with larger MFA. In the present paper, we show for the first time that this relation even exists for thin (200 μm) sections of wood, which provides strong evidence for the fact that the MFA optimizes the extensibility of wood. In a combination of tensile tests with structural investigations by small angle X-ray scattering on the same sample of Picea abies, we found a remarkable increase in maximum strain with increasing MFA, and also a change in the elastic moduli.

Zinc and mechanical prowess in the jaws of nereis a marine worm

Higher animals typically rely on calcification to harden certain tissues such as bones and teeth. Some notable exceptions can be found in invertebrates: The fangs, teeth, and mandibles of diverse arthropod species have been reported to contain high levels of zinc. Considerable quantities of zinc also occur in the jaws of the marine polychaete worm Nereis sp. High copper levels in the polychaete worm Glycera dibranchiata recently were attributed to a copper-based biomineral reinforcing the jaws. In the present article, we attempt to unravel the role of zinc in Nereis limbata jaws, using a combination of position-resolved state-of-the-art techniques. It is shown that the local hardness and stiffness of the jaws correlate with the local zinc concentration, pointing toward a structural role for zinc. Zinc always is detected in tight correlation with chlorine, suggesting the presence of a zinc–chlorine compound. No crystalline inorganic phase was found, however, and results from x-ray absorption spectroscopy further exclude the presence of simple inorganic zinc–chlorine compounds in amorphous form. The correlation of local histidine levels in the protein matrix and zinc concentration leads us to hypothesize a direct coordination of zinc and chlorine to the protein. A comparison of the role of the transition metals zinc and copper in the jaws of two polychaete worm species Nereis and Glycera, respectively, is presented.

Imaging of the helical arrangement of cellulose fibrils in wood by synchrotron X-ray microdiffraction

A complete image of the helical arrangement of cellulose fibrils in the S2 layer of adjacent wood cells of Picea abies (Norwegian spruce) was obtained by applying position-resolved synchrotron X-ray microdiffraction on cells in cross section. In contrast to conventional fiber diffraction studies, the incident beam was parallel to the longitudinal cell axis, resulting in a glancing angle μ far from 90° with respect to the cellulose fibrils. This special choice of diffraction geometry allowed us to take advantage of an asymmetry effect in the two-dimensional diffraction patterns arising from the curvature of the Ewald sphere to obtain information on the local orientation of the cellulose fibrils. The small size of the beam, smaller than the thickness of a single cell wall, allowed mesh scans over intact transverse sections of adjacent wood cells with a microscopic position resolution. The scan yielded a map of diffraction patterns that could readily serve as a microscopic image. Each of the diffraction patterns was then used to evaluate the local orientation of the cellulose fibrils at the actual beam position. The combination of these results gave an image of cellulose fibrils forming (Z) helices in several adjacent wood cells.

Heme-Solvent Coupling: A Mössbauer Study of Myoglobin in Sucrose

The Mössbauer effect of 57Fe-enriched samples was used to investigate the coupling of 80% sucrose/water, a protein-stabilizing solvent, to vibrational and diffusive modes of the heme iron of CO-myoglobin. For comparison we also determined the Mössbauer spectra of K4 57Fe (CN)6 (potassium ferrocyanide, PFC), where the iron is fully exposed in the same solvent. The temperature dependence of the Mössbauer parameters derived for the two samples proved to be remarkably similar, indicative of a strong coupling of the main heme displacements to the viscoelastic relaxation of the solvent. We show that CO escape out of the heme pocket couples to the same type of fluctuations, whereas intramolecular bond formation involves solvent-decoupled heme deformation modes that are less prominent in the Mössbauer spectrum. With respect to other solvents, however, sucrose shows a reduced viscosity effect on heme displacements and the kinetics of ligand binding due to preferential hydration of the protein. This result confirms thermodynamic predictions of the stabilizing action of sucrose by a dynamic method.

Cellulose microfibril angles in a spruce branch and mechanical implications

The nanostructure of the wood cell wall and, in particular the tilt angle of the cellulose fibrils versus the longitudinal cell axis (microfibril angle, MFA), are known to play a key role in determining the mechanical properties of wood. A variation of microfibril angles during growth may therefore be regarded as a means to adapt to different loading situations. In the present study, a branch of Norway spruce (Picea abies) was used as a model system. The change of microfibril angles with increasing age and size of the branch and therefore increasing gravitational load was systematically investigated. Small angle X-ray scattering (SAXS) was applied to obtain a map of MFA all over the branch as a function of the distance from the trunk within each annual ring. It was found that in compression wood the MFA decreased continuously from the trunk towards the tip in all annual rings. In opposite wood, however, the course of microfibril angles was found to change considerably with the age of the branch: in the outer annual rings, very small microfibril angles occurred in the middle part of the branch. The results are discussed in view of the mechanical implications of different microfibril angles.

Deformation and energy absorption of wood cell walls with different nanostructure under tensile loading

The nanostructure of the S2 cell wall layer in tracheids of Picea abies (Norwegian spruce), in particular the cellulose microfibril angle, has been shown to control not only the stiffness but also the extensibility of wood within a wide range. In order to further elucidate this effect, the deformation of wood under tensile load parallel to the longitudinal cell axis was studied in a contact-free way using a video extensometer. The combination of these measurements with small-angle X-ray scattering on the same microtome sections allowed us to establish a direct relationship between the microfibril angle and deformation behaviour. The microfibril angle was shown to influence not only the extensibility in longitudinal direction but also the deformation perpendicular to the applied load. Moreover, the results showed that the energy absorption capacity is higher for specimens with larger microfibril angle. SEM pictures of the fractured samples indicated clearly the differences in the fracture process as the fracture zones of samples with low microfibril angle were smooth and the fracture zones of samples with high microfibril angle were heavily torn and deformed indicating a more ductile behaviour.

Halogenated Veneers: Protein Cross-Linking and Halogenation in the Jaws of Nereis ,a Marine Polychaete Worm

Mineralized tissues are produced by most living organisms for load and impact functions. In contrast, the jaws of the clam worm, Nereis, are hard without mineralization. However, they are peculiarly rich in halogens, which are associated with a variety of post‐translationally modified amino acids, many of which are multiply halogenated by chlorine, bromine, and/or iodine. Several of these modified amino acids, namely dibromohistidine, bromoiodohistidine, chloroiodotyrosine, bromoiodotyrosine, chlorodityrosine, chlorotrityrosine, chlorobromotrityrosine, and bromoiodotrityrosine, have not been previously reported. We have found that the distributions of Cl, Br, and I differ: Cl is widespread whereas Br and I, although not colocalized, are concentrated in proximity to the external jaw surfaces. By using nanoindentation, we show that Br and I are unlikely to play a purely mechanical role, but that the local Zn and Cl concentrations and jaw microstructure are the prime determinants of local jaw hardness. Several of the post‐translationally modified amino acids are akin to those found in various sclerotized structures of invertebrates, and we propose that they are part of a cross‐linked protein casing.

Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins.

ABSTRACT Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.

The importance of seasonal differences in the cellulose microfibril angle in softwoods in determining acoustic properties

The influence of the micro- and mesoscopic structure of wood cell walls on the acoustic properties of softwood was investigated in a synchrotron X-ray microbeam diffraction experiment with particular attention to the seasonal differences in crystallographic features. A multiple regression analysis was performed for data from 12 different softwood species in order to determine the dependence of longitudinal relative Youngs modulus (E/ρ) and loss tangent (tanδ) on seasonal cellulose microfibril angles (MFAs), crystal width of cellulose microfibrils etc. We conclude that a low MFA in both latewood and earlywood yields high E/ρ and low tanδ, which is an attribute of wood used as violin or piano soundboards. Among the softwood species we characterized Sitka spruce best fits this criterion.

The internal structure of single carbon fibers determined by simultaneous small- and wide-angle X-ray scattering

Simultaneous small-angle scattering and wide-angle diffraction using a synchrotron radiation microbeam was applied for the first time to investigate single carbon fibers in a position-resolved way. Taking into account the exact X-ray beam profile and examining the fibers in two scattering geometries allowed a discrimination between different models for the internal arrangement of carbon layers and pores. For a fiber based on polyacrylnitrile the carbon layers were randomly oriented within the fiber cross section, whereas in a mesophase-pitch based fiber the layers were arranged in a radial structure.