Action of AA9 lytic polysaccharide monooxygenase enzymes on different cellulose allomorphs.

Lytic polysaccharide monooxygenase (LPMO)-catalyzed oxidative processes play a major role in natural biomass conversion. Despite their oxidative cleavage at the surface of polysaccharides, understanding of their mode of action, and the impact of structural patterns of the cellulose fiber on LPMO activity is still not fully understood. In this work, we investigated the action of two different LPMOs from Podospora anserina on celluloses showing different structural patterns. For this purpose, we prepared cellulose II and cellulose III allomorphs from cellulose I cotton linters, as well as amorphous cellulose. LPMO action was monitored in terms of surface morphology, molar mass changes and monosaccharide profile. Both PaLPMO9E and PaLPMO9H were active on the different cellulose allomorphs (I, II and III), and on amorphous cellulose (PASC) whereas they displayed a different behavior, with a higher molar mass decrease observed for cellulose I. Overall, the pretreatment with LPMO enzymes clearly increased the accessibility of all types of cellulose, which was quantified by the higher carboxylate content after carboxymethylation reaction on LPMO-pretreated celluloses. This work gives more insight into the action of LPMOs as a tool for deconstructing lignocellulosic biomass to obtain new bio-based building blocks.

Xyloglucan-Cellulose Nanocrystals Mixtures: A Case Study of Nanocolloidal Hydrogels and Levers for Tuning Functional Properties.

The development of fully biobased hydrogels obtained by simple routes and in the absence of toxic or environmentally harmful reagents is a major challenge in meeting new societal demands. In this work, we discuss the development of hydrogels made from cellulose nanocrystals (CNCs) and xyloglucan (XG), two non-toxic, renewable, and biobased components. We present three strategies to fine-tune the functional properties. The first one consists in varying the XG/CNC ratio that leads to the modulation of the mechanical properties of hydrogels as well as a better comprehension of the gel mechanism formation. The second relies on tuning the XG chains' interaction by enzymatic modification to achieve thermoresponsive systems. Finally, the third one is based on the increase in the hydrogel solid content by osmotic concentration. The high-solid-content gels were found to have very high mechanical properties and self-healing properties that can be used for molding materials. Overall, these approaches are a case study of potential modifications and properties offered by biobased nanocolloidal hydrogels.

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Flexoelectric and piezoelectric effects in micro- and nanocellulose films.

In this work, we evaluated the flexoelectric and piezoelectric contributions to the overall macroscopic polarization in cellulose films. To this end, the flexoelectric µ31 and transverse effective piezoelectric e31,f coefficients of cellulose films were determined using cantilever beam bending. The experiments were based on theoretical developments allowing to separate the flexoelectric from the piezoelectric contribution, represented by an effective flexoelectric coefficient, µeff, depending on both e31,f and µ31. Five free-standing and stainless steel/cellulose bilayer films were prepared from cellulose showing different morphologies and surface charge degrees: two almost neutral cellulose microfibers (CMF) and three (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidized cellulose micro- (TCMF) and nanofibers (TCNF) bearing negative charged groups on the surface. The dielectric properties of the films indicated a low dielectric constant for unmodified CMF, and a huge increase for TEMPO-oxidized samples, which were up to 9 times higher than poly(vinylidene fluoride)-based polymers. TEMPO-oxidized cellulose films exhibited the largest flexoelectric coefficients (almost 7 times higher than those of synthetic polymer dielectrics), which evidenced that the presence of polar groups and surface charge boosted both flexoelectricity and piezoelectricity in unpoled cellulose films. These findings pave the way towards sustainable cellulose-based curvature sensors with large effective flexoelectric coefficients, without the need of preliminary energy consuming poling step.

Optimized Lytic Polysaccharide Monooxygenase Action Increases Fiber Accessibility and Fibrillation by Releasing Tension Stress in Cellulose Cotton Fibers.

Lytic polysaccharide monooxygenase (LPMO) enzymes have recently shaken up our knowledge of the enzymatic degradation of biopolymers and cellulose in particular. This unique class of metalloenzymes cleaves cellulose and other recalcitrant polysaccharides using an oxidative mechanism. Despite their potential in biomass saccharification and cellulose fibrillation, the detailed mode of action of LPMOs at the surface of cellulose fibers still remains poorly understood and highly challenging to investigate. In this study, we first determined the optimal parameters (temperature, pH, enzyme concentration, and pulp consistency) of LPMO action on the cellulose fibers by analyzing the changes in molar mass distribution of solubilized fibers using high performance size exclusion chromatography (HPSEC). Using an experimental design approach with a fungal LPMO from the AA9 family (PaLPMO9H) and cotton fibers, we revealed a maximum decrease in molar mass at 26.6 °C and pH 5.5, with 1.6% w/w enzyme loading in dilute cellulose dispersions (100 mg of cellulose at 0.5% w/v). These optimal conditions were used to further investigate the effect of PaLPMO9H on the cellulosic fiber structure. Direct visualization of the fiber surface by scanning electron microscopy (SEM) revealed that PaLPMO9H created cracks on the cellulose surface while it attacked tension regions that triggered the rearrangement of cellulose chains. Solid-state NMR indicated that PaLPMO9H increased the lateral fibril dimension and created novel accessible surfaces. This study confirms the LPMO-driven disruption of cellulose fibers and extends our knowledge of the mechanism underlying such modifications. We hypothesize that the oxidative cleavage at the surface of the fibers releases the tension stress with loosening of the fiber structure and peeling of the surface, thereby increasing the accessibility and facilitating fibrillation.

Unraveling the control of reversibility for actuators based on cellulose nanofibers.

In this work, we have prepared cellulose-based actuators taking advantage of the pH-sensitive solubility of chitosan (CH) and the mechanical strength of CNFs. Bilayer films were prepared by vacuum filtration inspired by plant structures that exhibit reversible deformation under pH changes. The presence of CH in one of the layers led to asymmetric swelling at low pH, thanks to the electrostatic repulsion between charged amino groups of CH, and the subsequent twisting with the CH layer on the outside. Reversibility was achieved by substituting pristine CNFs with carboxymethylated CNFs (CMCNFs), that are charged at high pH and thus competed with the effects of amino groups. Swelling and mechanical properties of layers under pH changes were studied by gravimetry and dynamic mechanical analysis (DMA) to quantify the contribution of chitosan and the modified CNFs on the reversibility control. This work evidenced the key role of surface charge and layer stiffness to achieve reversibility. Bending was triggered by the different water uptake of each layer, and shape recovery was achieved when the shrunk layer shower higher rigidity than the swollen layer.

Adsorption of apple xyloglucan on cellulose nanofiber depends on molecular weight, concentration and building blocks.

Apple xyloglucan (XyG) structural features (molecular weight and building motifs) were studied in relation with the hemicellulose interaction with cellulose nanofiber (CNF) through adsorption isotherm, high performance size exclusion chromatography and high-performance anion exchange chromatography analyses. Up to 75.6 µg of apple XyG adsorbed/mg of CNF. XyG adsorption depended on both Mw and XyG/CNF ratio. The adsorption capacity increased with Mw irrespectively of XyG/CNF ratio. At XyG/CNF ratio > 0.05, the adsorption of high Mw populations (2.0 × 105, 7.9 × 104 g mol-1) gradually ceased, while it kept increasing for low Mw populations (2.6 × 104, 1.2 × 104 g mol-1). The unbranched glucan segments and the galactosylated XLLG motif showed the highest binding affinity to CNF. Most of the XLLG motif was found in "loops and tails", whereas most of the XyG unbranched glucan segments interact directly with CNF.

Xyloglucan-cellulose nanocrystal-chitosan double network hydrogels for soft actuators.

Hydrogels are materials consisting in a three-dimensional hydrophilic polymer network swollen by a large amount of water. An efficient strategy to elaborate hydrogels consists in establishing double polymer networks in order to achieve high strengthening effect associated with other properties such as transparency or tailored swelling capacities. In this work, we prepared cellulose nanocrystals (CNC)-based hydrogels with double network architecture. The first network, formed by CNC and xyloglucan (XG), takes advantage of entropic adsorption of XG on the CNC surface while the second network relies on electrostatic interactions between cationic Chitosan (Chi) and anionic CNC. Hydrogels with different compositions were successfully prepared. Their rheological properties, stability and swelling capacities in acidic and alkaline solutions were evaluated. Internal organizations of hydrogels were investigated by fluorescence microscopy after polymer labelling and polarized optical microscopy (POM). Finally, hydrogels demonstrated excellent mechanical properties and tuneable swelling capacities that can be leveraged for the implementation of bilayer actuators. Therefore, we further prepared films composed of two hydrogels layers, each one containing a different XG/CNC ratio. Bilayered films bended in water due to the asymmetric swelling of layers and the extent of bending can be modulated by the XG/CNC ratio.

Dextrans and dextran derivatives as polyelectrolytes in layer-by-layer processing materials - A review.

The layer-by-layer technique (LbL) using polysaccharides is receiving increasing interest as the preparation of nano- and micro- multilayer objects composed by natural materials is a promising alternative for actual systems. The use of bacterial polysaccharides and more particularly, dextrans and dextran derivatives, in LbL assemblies allows the simple formation of biodegradable and biocompatible multilayers with engineered functionalities. The choice of dextrans and derivatives along with the assembly conditions can indeed control internal structure and physical, chemical and biological properties. In this review, we describe the use of dextrans and dextran derivatives into multilayers presented in literature, from the method and principles to the key parameters that need to be considered in their formations. We present their valorisation into nanoobjects with specific and stimuli-responsive properties that are mainly exploited for biomedical applications as drug delivery devices.

Divergent growth of poly(amidoamine) dendrimer-like branched polymers at the reducing end of cellulose nanocrystals.

This paper presents the growth of dendritic polymers at the reducing ends of cellulose nanocrystals by the "grafting from" approach. We took advantage of the chemically differentiated ends of cellulose nanocrystals to specifically synthesize dendrimers at their reducing end by the divergent approach. We used acid-amine coupling reactions in aqueous media to synthesize the carboxylic acid- or amine-terminated poly(amidoamine) dendrimers. The growth of dendrimer generations was monitored by UV and FTIR spectroscopies, and we successfully introduced up to 4 generations. The dendrimer growth at reducing ends was demonstrated by the nanocrystal adsorption driven by the peripheral amino groups onto gold surfaces. Hence, the results from quartz crystal microbalance with dissipation (QCM-D) pointed to a rather upright orientation of the dendrimer-modified cellulose nanocrystals. As the generation increased, the adsorbed layers appeared to be more flexible, which demonstrated that the functionality at the reducing end can successfully tune the properties of cellulose nanocrystals.

Bibliometric survey and network analysis of biomimetics and nature inspiration in engineering science.

The field encompassing biomimetics, bioinspiration and nature inspiration in engineering science is growing steadily, pushed by exogenous factors like the search for potentially sustainable engineering solutions that might already exist in nature. With the help of information provided by a bibliometric database and further processed with a dynamic network and semantic analysis tool, we provide insight at two scales into the corpus of nature-inspired engineering field and its dynamics. At the macroscale, the Web of Science®(WoS) categories, countries and institutions are ranked and ordered by thematic clusters and country networks, highlighting the leading countries and institutions and how they focus on specific topics. Such an insight provides an overview at the macroscale that can be valuable to orient scientific strategy at the country level. At the mesoscale, where science is incarnated by collaborative networks of authors and institutions that run across countries, we identify six semantic clusters and subclusters within them, and their dynamics. We also pinpoint leading academic collaborative networks and their activity in relation to the six semantic clusters. Trends and prospective are also discussed. Typically, one observes that the field is becoming mature since, starting by imitating nature, it proceeded with mimicking more complex natural structures and functions and now it investigates ways used in nature in response to changes in the environment and implements them in innovative and adaptive artefacts. The sophistication of devices, methods and tools has been increasing over the years as well as their functionalities and adaptability, whereas the size of devices has decreased at the same time.

Dextran-based polyelectrolyte multilayers: Effect of charge density on film build-up and morphology.

We have studied the growth process of thin polyelectrolyte (PE) films fabricated by the layer-by-layer assembly (LbL) and composed of Dextran sulfate with high (DexS H) and low (DexS L) sulfation rate and poly(allylamine hydrochloride) (PAH). Film growths were monitored by combining Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM). Even though, the two films growth up to 10 bilayers, QCM-D showed that polyelectrolyte pairs do not display similar behaviours. (PAH/DexS H) systems lead to linear growth, i.e. amounts deposited increase both for PAH and DexS H, while the PAH/DexS L pair generated zig-zag shaped asymmetric growth. Film water contents were determined by QCM-D solvent exchange and SPR experiments. DexS L contains less water than DexS H and in agreement with the QCM-D dissipation values that suggest the formation of more rigid films in the case of DexS L than DexS H. Surface morphology investigated by AFM display distinct surface patterns since DexS H form thin films with fibril-like morphology covering all the surface while heterogeneous films with "puddle-like" aggregates were imaged in the case of DexS L. Difference of charge compensation and charge neutralisation between both systems likely lead to dissimilar growth mechanisms that are tentatively proposed in this paper.

Cellulose Nanocrystal-Fibrin Nanocomposite Hydrogels Promoting Myotube Formation.

Cellulose nanocrystals (CNCs) have been widely studied as fillers to form reinforced nanocomposites with a wide range of applications, including the biomedical field. Here, we evaluated the possibility to combine them with fibrinogen and obtain fibrin hydrogels with improved mechanical stability as potential cellular scaffolds. In diluted conditions at a neutral pH, it was evidenced that fibrinogen could adsorb on CNCs in a two-step process, favoring their alignment under flow. Composite hydrogels could be prepared from concentrated fibrinogen solutions and nanocrystals in amounts up to 0.3 wt %. CNCs induced a significant modification of the initial fibrin fibrillogenesis and final fibrin network structure, and storage moduli of all nanocomposites were larger than those of pure fibrin hydrogels. Moreover, optimal conditions were found that promoted muscle cell differentiation and formation of long myotubes. These results provide original insights into the interactions of CNCs with proteins with key physiological functions and offer new perspectives for the design of injectable fibrin-based formulations.

Development of Bio-Inspired Hierarchical Fibres to Tailor the Fibre/Matrix Interphase in (Bio)composites.

Several naturally occurring biological systems, such as bones, nacre or wood, display hierarchical architectures with a central role of the nanostructuration that allows reaching amazing properties such as high strength and toughness. Developing such architectures in man-made materials is highly challenging, and recent research relies on this concept of hierarchical structures to design high-performance composite materials. This review deals more specifically with the development of hierarchical fibres by the deposition of nano-objects at their surface to tailor the fibre/matrix interphase in (bio)composites. Fully synthetic hierarchical fibre reinforced composites are described, and the potential of hierarchical fibres is discussed for the development of sustainable biocomposite materials with enhanced structural performance. Based on various surface, microstructural and mechanical characterizations, this review highlights that nano-objects coated on natural fibres (carbon nanotubes, ZnO nanowires, nanocelluloses) can improve the load transfer and interfacial adhesion between the matrix and the fibres, and the resulting mechanical performances of biocomposites. Indeed, the surface topography of the fibres is modified with higher roughness and specific surface area, implying increased mechanical interlocking with the matrix. As a result, the interfacial shear strength (IFSS) between fibres and polymer matrices is enhanced, and failure mechanisms can be modified with a crack propagation occurring through a zig-zag path along interphases.

Cellulose Nanofibrils/Xyloglucan Bio-Based Aerogels with Shape Recovery.

Bio-based aerogels containing cellulose nanofibrils (CNFs) are promising materials due to the inherent physical properties of CNF. The high affinity of cellulose to plant hemicelluloses (xyloglucan, xylan, pectin) is also an opportunity to develop biomaterials with new properties. Here, we prepared aerogels from gelled dispersions of CNFs and xyloglucan (XG) at different ratios by using a freeze-casting procedure in unidirectional (UD) and non-directional (ND) manners. As showed by rheology analysis, CNF and CNF/XG dispersions behave as true gels. We investigated the impact of the freezing procedure and the gel's composition on the microstructure and the water absorption properties. The introduction of XG greatly affects the microstructure of the aerogel from lamellar to cellular morphology. Bio-based aerogels showed high water absorption capacity with shape recovery after compression. The relation between morphology and aerogel compositions is discussed.

Hierarchical thermoplastic biocomposites reinforced with flax fibres modified by xyloglucan and cellulose nanocrystals.

This work is focused on the modification of the interphase zone in short flax fibres / polypropylene (PP) composites by a bio-inspired modification of fibres called "nanostructuration" that uses the adsorption of biomass by-products, i.e. cellulose nanocrystals (CNC) and xyloglucan (XG), to create hierarchical flax fibres. The wettability and interfacial adhesion study reveals a strong decrease in the polar character of CNC modified flax fibres, hence increasing the work of adhesion with PP. Moreover, combining XG/CNC modified interphases with MAPP coupling agent enhances the ultimate mechanical properties of biocomposites with higher tensile strength and work of rupture, and modifies failure mechanisms as revealed by in situ micro-mechanical tensile SEM experiments. Bio-based hierarchical composites inspired by naturally occurring nanostructures open a new path for the development of sustainable composites with enhanced structural properties.

Bioinspired Thermoresponsive Xyloglucan-Cellulose Nanocrystal Hydrogels.

Thermoresponsive hydrogels present unique properties, such as tunable mechanical performance or changes in volume, which make them attractive for applications including wound healing dressings, drug delivery vehicles, and implants, among others. This work reports the implementation of bioinspired thermoresponsive hydrogels composed of xyloglucan (XG) and cellulose nanocrystals (CNCs). Starting from tamarind seed XG (XGt), thermoresponsive XG was obtained by enzymatic degalactosylation (DG-XG), which reduced the galactose residue content by ∼50% and imparted a reversible thermal transition. XG with native composition and comparable molar mass to DG-XG was produced by an ultrasonication treatment (XGu) for a direct comparison of behavior. The hydrogels were prepared by simple mixing of DG-XG or XGu with CNCs in water. Phase diagrams were established to identify the ratios of DG-XG or XGu to CNCs that yielded a viscous liquid, a phase-separated mixture, a simple gel, or a thermoresponsive gel. Gelation occurred at a DG-XG or XGu to CNC ratio higher than that needed for the full surface coverage of CNCs and required relatively high overall concentrations of both components (tested concentrations up to 20 g/L XG and 30 g/L CNCs). This is likely a result of the increase in effective hydrodynamic volume of CNCs due to the formation of XG-CNC complexes. Investigation of the adsorption behavior indicated that DG-XG formed a more rigid layer on CNCs compared to XGu. Rheological properties of the hydrogels were characterized, and a reversible thermal transition was found for DG-XG/CNC gels at 35 °C. This thermoresponsive behavior provides opportunities to apply this system widely, especially in the biomedical field, where the mechanical properties could be further tuned by adjusting the CNC content.

Influence of arabinoxylan on the drying of cellulose nanocrystals suspension: From coffee ring to Maltese cross pattern and application to enzymatic detection.

Evaporation of sessile droplet containing suspension of cellulose nanocrystals (CNC) results on birefringent coffee ring pattern (CR), due to the concentration increase and self-assembly of CNC carried by the flow at the edge of evaporating droplet. In this work, we studied the apparition of Maltese cross pattern, (MC) after addition of an hydrosoluble biopolymer belonging to the hemicellulose family, i.e. arabinoxylan (AX). To investigate the mechanisms that control MC pattern apparition, distribution of the two components inside the dried droplet was investigated using FTIR. CNC and AX were found to be homogenously deposited and CNC self-assembly induces nanoparticles orientation in the CR deposit. We demonstrate that the increase of concentration during drying induces gelation of CNC/AX mixture leading to MC pattern apparition. We take advantage of the apparition of MC pattern to develop a novel catalytic activity detection assay based on the variation of viscosity. Indeed, addition of Endo-1,4-ß-Xylanase (Xyl) addition to a suspension containing CNC/AX complex leads to hydrolysis of AX that decrease in droplet viscosity leading to MC disappearance. The enzymatic detection assay is thus simple, easy to handle, fast, sensitive and do not require complex analytical devices.

Asymmetric modification of cellulose nanocrystals with PAMAM dendrimers for the preparation of pH-responsive hairy surfaces.

In this work, we present a straightforward method to attach a globular dendrimer at the reducing end of cellulose nanocrystals obtained from tunicates (t-CNC). We investigated the first four generations of poly(amidoamine) dendrimers (PAMAMs G0 to G3) to obtain hybrid t-CNCs. The aggregation behavior of hybrid t-CNCs was studied by dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM); and interactions of these asymmetric nanoparticles with gold surface were elucidated using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR). Hybrid t-CNCs formed hairy layers onto gold surface combining the properties of rigid rod-like nanocrystals with globular and flexible PAMAM dendrimers. Moreover, the presence of amino groups provided pH-responsive properties to hybrid t-CNCs, and QCM-D results revealed reversible swelling/deswelling behavior. Thereby, we achieved to synthesize tree-shaped functional bio-based materials that adsorbed on gold and formed pH-responsive hairy surfaces.