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International audience; This work presents a dynamic view of the enzymatic degradation of maize cell walls, and sheds new light on the recalcitrance of hot water pretreated maize stem internodes. Infra-red microspectrometry, mass spectrometry, fluorescence recovery after photobleaching and fluorescence imaging were combined to investigate enzymatic hydrolysis at the cell scale. Depending on their polymer composition and organisation, cell types exhibits different extent and rate of enzymatic degradation. Enzymes act sequentially from the cell walls rich in accessible cellulose to the most recalcitrant cells. This phenomenon can be linked to the heterogeneous distribution of enzymes in the liquid medium and the adsorption/desorption mechanisms that differ with the type of cell.

International audience; This work proposes a multi-scale study of the properties of leaf sheath date palm fibres currently considered as agricultural waste. Firstly, by using optical and electronic microscopy, two main types of bundles were identified which have profoundly different structures. Biochemical analysis and X-ray diffraction (XRD) revealed a low degree of crystallinity but a significant lignin content of about 17% giving the bundles a very cohesive structure as well as a good thermal stability in addition to a singular behaviour in dynamic vapour sorption. An average cell wall stiffness in the order of 16 GPa was highlighted by Atomic Force Microscopy in mechanical mode but tensile tests on bundles have revealed low stiffness and strength but a high elongation. These results combined with the cellular structure of these bundles, provides the potential of these wastes as cost effective and environmentally friendly composite reinforcements for high energy absorption and improved acoustics functions. (C) 2017 Elsevier Ltd. All rights reserved.

International audience; In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently.[br/] Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images.[br/] Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

The conversion of cell wall biomass to sugar for fermentation to ethanol requires chemical or physical pretreatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Multiscale study of biomass deconstruction gives access to key insights into the cell wall and lignin changes induced by pretreatment. Few studies have compared the effect of pretreatment of different biomass species on the cell wall accessibility. Considering representative softwood (pine) and hardwood (poplar) biomass, we studied the impact of two pretreatments on enzymatic saccharification and cell wall structure and accessibility. Lignin fluorescence properties were investigated by measuring fluorescence lifetime in addition to chemical analysis, and the accessibility of biomass was assessed using fluorescent probes consisting of rhodamine labeled polyethylene glycol (PEG) molecules ranging from 10 to 40 kDa. Hot water treatment and chlorite delignification altered chemical structure and fluorescence lifetime, which was positively correlated with glucose conversion and negatively correlated with lignin and β-O-4′ contents. Imaging distribution of the probes indicated that chlorite pretreatment resulted in a more uniform distribution of probe in the cell wall compared to hot water treatment. The interaction between cell wall and fluorescent PEG probes was evaluated using Forster Resonance Energy Transfer (FRET) and fluorescence microscopy. The FRET efficiency showed a high negative correlation with the probe size and was greatly increased by chlorite delignification, reflecting increased accessibility to the probe and interaction. Thus the accessibility and interactions of small probes in pretreated biomass could be a relevant indicator of potential for saccharification, whereas fluorescence lifetime provides a new criteria for assessing relevant cell wall structural modifications related to enzymatic conversion of lignocellulosic biomass.

Background Biomass recalcitrance to enzymatic hydrolysis has been assigned to several structural and chemical factors. However, their relative importance remains challenging to evaluate. Three representative biomass species (wheat straw, poplar and miscanthus) were submitted to four standard pretreatments (dilute acid, hot water, ionic liquid and sodium chlorite) in order to generate a set of contrasted samples. A large array of techniques, including wet chemistry analysis, porosity measurements using NMR spectroscopy, electron and fluorescence microscopy, were used in order to determine possible generic factors of biomass recalcitrance. Results The pretreatment conditions selected allowed obtaining samples displaying different susceptibility to enzymatic hydrolysis (from 3 up to 98% of the initial glucose content released after 96 h of saccharification). Generic correlation coefficients were calculated between the measured chemical and structural features and the final saccharification rates. Increases in porosity displayed overall strong positive correlations with saccharification efficiency, but different porosity ranges were concerned depending on the considered biomass. Lignin-related factors displayed highly negative coefficients for all biomasses. Lignin content, which is likely involved in the correlations observed for porosity, was less detrimental to enzymatic hydrolysis than lignin composition. Lignin influence was highlighted by the strong negative correlation with fluorescence intensity which mainly originates from monolignols in mature tissues. Conclusions Our results provide a better understanding of the factors responsible for biomass recalcitrance that can reasonably be considered as generic. The correlations with specific porosity ranges are biomass species-dependent, meaning that enzymes cocktails with fitted enzyme size are likely to be needed to optimise saccharification depending on the biomass origin. Lignin composition, which probably influences its structure, is the most important parameter to overcome to enhance enzymes access to the polysaccharides. Accordingly, fluorescence intensity was found to be a rapid and simple method to assess recalcitrance after pretreatment. Electronic supplementary material The online version of this article (10.1186/s13068-018-1053-8) contains supplementary material, which is available to authorised users.

International audience; Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery. Plant cell walls constitute the largest renewable source of biomass on Earth that can supply environmental benefits for the production of fuel, chemicals and materials. They are composed by lignocellulose made from three main polymers (cellulose, hemicelluloses, lignin) assembling as a network whose structural and chemical complexity hampers hydrolysis of cellulose by enzymes and microorganisms 1. In addition, cell walls display high variability depending on genetic and environmental factors, as well as plant tissue and cell types 2–4. Understanding plant cell wall complexity during lignocellulosic bioconversion is therefore important to identify critical features impacting hydrolysis, for optimising pretreatments of biomass 5 and enzyme cocktails 6. Investigation of the dynamics of lignocellulose hydrolysis requires physicochemical characterization and mul-tiscale visualization 7,8. Combined approach using multiple microscopic techniques including scanning electron microscopy, atomic force microscopy, light microscopy, immunocytochemistry and microspectrometry can be used to monitor microstructural and topochemical heterogeneity of plant cell walls and their recalcitrance at tissue, cell and subcellular levels 9–15. In addition, spatial and temporal imaging of enzymes distribution within lignocellulose substrates can be carried out by means of immunoprobe or fluorescent labelled protein 16–18 to study accessibility at the molecular scale 19,20. However real time imaging of cell wall microstructure and enzyme distribution during bioconversion still remains challenging. Enzymatic degradation of cellulose and hemicelluloses involves several types of enzymes, namely glycoside hydrolases that work synergistically 21,22. To overcome the recalcitrance of plant polysaccharides, filamentous fungi and bacteria secrete lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of gly-coside bonds 23–25. In industrial processes, addition of LPMOs to cellulolytic cocktails leads to the reduction of the enzyme loading required for efficient saccharification of lignocellulosic biomass 26. These powerful enzymes are copper-containing enzymes classified within the auxiliary activity (AA) class of the CAZy database (www.cazy. org 27) in AA9-AA11 and AA13 families. AA9 LPMOs are mostly active on cellulose but some have been shown to act on xyloglucan and glucomannan 25,28. Despite their recognized boosting effect on biomass hydrolysis, AA9 LPMOs activity has been essentially investigated on model substrates with only sparse studies focusing on the insoluble fraction of the substrate that show their disruptive action at the surface of cellulosic fibers 29–31. To our

International audience; Interest in hemp (Cannabis sativa L.) is increasing due to the development of a new range of industrial applications based on bast fibers. However the variability of bast fiber yield and quality represents an important barrier to further exploitation. Primary and secondary fiber content was examined in two commercial hemp varieties (Fedora 17, Santhica 27) grown under contrasted sowing density and irrigation conditions. Both growing conditions and hemp varieties impact stem tissue architecture with a large effect on the proportion of secondary fibers but not primary fibers. Attenuated total reflectance infrared spectroscopy allowed the discrimination of manually-isolated native primary fibers and secondary fibers but did not reveal any clustering according to growing conditions and variety. Infrared data were confirmed by wet chemistry analyses that revealed slight but significant differences between primary and secondary fiber cell wall composition. Infrared spectroscopy of technical fibers obtained after mechanical defibering revealed differences with native primary, but not secondary fibers and also discriminated samples obtained from plants grown under different conditions. Altogether the results suggested that the observed variability of hemp technical fibers could be partially explained by i) differences in secondary fiber production and ii) differential behavior during mechanical defibering resulting in unequal separation of primary and secondary fibers.

Background: The lignocellulosic cell wall network is resistant to enzymatic degradation due to the complex chemical and structural features. Pretreatments are thus commonly used to overcome natural recalcitrance of lignocellulose. Characterization of their impact on architecture requires combinatory approaches. However, the accessibility of the lignocellulosic cell walls still needs further insights to provide relevant information. Results: Poplar specimens were pretreated using different conditions. Chemical, spectral, microscopic and immunolabeling analysis revealed that poplar cell walls were more altered by sodium chlorite-acetic acid and hydrothermal pretreatments but weakly modified by soaking in aqueous ammonium. In order to evaluate the accessibility of the pretreated poplar samples, two fluorescent probes (rhodamine B-isothiocyanate-dextrans of 20 and 70 kDa) were selected, and their mobility was measured by using the fluorescence recovery after photobleaching (FRAP) technique in a full factorial experiment. The mobility of the probes was dependent on the pretreatment type, the cell wall localization (secondary cell wall and cell corner middle lamella) and the probe size. Overall, combinatory analysis of pretreated poplar samples showed that even the partial removal of hemicellulose contributed to facilitate the accessibility to the fluorescent probes. On the contrary, nearly complete removal of lignin was detrimental to accessibility due to the possible cellulose-hemicellulose collapse. Conclusions: Evaluation of plant cell wall accessibility through FRAP measurement brings further insights into the impact of physicochemical pretreatments on lignocellulosic samples in combination with chemical and histochemical analysis. This technique thus represents a relevant approach to better understand the effect of pretreatments on lignocellulose architecture, while considering different limitations as non-specific interactions and enzyme efficiency.

International audience; Peak-Force Quantitative Nano-Mechanical property mapping (PF-QNM) and Micro Tomography were applied to explore the architecture and nano-mechanical properties of hemp secondary fibres cell walls. After a comparative morphological analysis of both primary and secondary fibres, secondary bundles structure and mechanical properties were investigated. As a first, it was showed that, in spite of important morphological differences, secondary fibres stiffness was the same that primary fibres one; moreover, innovative tomographic images gave an inedited representation of secondary bundles confirming the intrusive development of secondary hemp fibres.

The hygroscopic properties of lignocellulose-based films were investigated by designing bio-inspired nanostructured assemblies of increasing complexity. Binary and ternary films were prepared from three main secondary cell wall polymers, namely, cellulose, hemicellulose (xylan or glucomannan) and lignin. The effect of lignin was studied by varying the manner of lignin introduction as the dehydropolymer (DHP, model lignin). The infrared analysis and water-sorption properties of the films that resulted from mixing DHP and polysaccharides were compared with films obtained from the polymerisation of coniferyl alcohol (lignin monomer) in the presence of polysaccharides. Comparison of the film behaviours was achieved using the GAB and PARK models for the sorption isotherms and the PEK model for the sorption kinetics. Our data indicated that without chemical functionalization, the water-sorption properties of the hemicellulose films were enhanced after forming lignin under mild enzymatic conditions. Notably, the hydrophobic nature of lignin was not expressed in these films, in contrast to the systems that resulted from a simple mixing of lignin with hemicelluloses. The presence of cellulose also modified these sorption properties, most likely by altering the interactions between the polymers and/or the monolignol reactions.

Mid-infrared (MIR) and near-infrared (NIR) spectroscopy provide useful information on the molecular composition of biological systems. Because they are sensitive to organic and mineral components, there is a growing interest in these techniques for the development of biomarkers that reflect intrinsic characteristics of plants and their mode of degradation. Due to their complexity and complementary nature, an important challenge is the combining of MIR and NIR information to identify discriminating wavenumbers in each wavenumber region, with the ultimate goal of assessing the biodegradation process of a lignocellulosic biomass at different time scales. This work investigates the potential of using the outer product to combine MIR and NIR spectra to highlight the connections between fundamental molecular vibrations and their combinations and bonds. Because this operation yields high-dimensional spectra, we propose to use a genetic algorithm to select the most discriminant wavenumbers within the degradation process. The results from two lignocellulosic biomasses with different biodegradation kinetics, miscanthus aerial parts and maize roots, confirm that the outer product combination of MIR and NIR spectral information allows a better discrimination of the biodegradation kinetic compared with the simple concatenation of MIR and NIR spectra or with the use of MIR or MIR spectral information separately. We show that the genetic algorithm selects wavenumbers that correspond to principal vibrations of chemical functional groups of compounds that undergo degradation/conversion during the biodegradation of the lignocellulosic biomass.

In addition to lignin, grass cell walls are characterized by the presence of hydroxycinnamic acids that play a significant role in cross-linking polymers into a cohesive network, and pretreatments are required to overcome the recalcitrance of lignocelluloses prior to enzymatic bioconversion of polysaccharides. The effects of dilute acid and ammonium hydroxide pretreatments were studied on the chemical composition and enzymatic saccharification of Miscanthus internodes fragments. The hydroxycinnamic acid content was reduced after both pretreatments, while lignin got enriched in condensed linked structures. In addition, dilute acid pretreatment was effective in decreasing xylan content of Miscanthus, while ammonia treatment induced a marked swelling effect on the cell walls of parenchyma, vascular sclerenchyma, and epidermal sclerenchyma. The phenol distribution at the cell level was estimated using UV transmission microspectrophotometry. Internode cell walls displayed different UV spectra according to the cell type. However, the secondary cell walls had similar UV spectra after pretreatment, whereas spectra recorded at the cell corner region displayed variations according to cell type and pretreatment. Acid pretreatment was more efficient than ammonia to improve the conversion of polysaccharides by a Trichoderma cellulolytic cocktail. Although pretreatments achieved moderate saccharification yields, the secondary cell walls were altered at some pit regions of the vascular sclerenchyma whereas parenchyma appeared recalcitrant. Variations in the UV spectra of enzyme-digested cell walls suggest pretreatment-dependent heterogeneity of the phenolic distribution in the more recalcitrant cell walls.

International audience; Interfacial affinity between lignin model compound (dehydrogenation polymer (DHPI) and cellulose nanocristals (CN) was studied before building a nanocomposite cellulose/lignin in multilayer form by spin-coating method. The adsorption isotherm of DHP was measured by ellipsometry at the liquid/CN film interface and showed that the surface concentration of adsorbed DHP increases with the bulk concentration in solution. The DHP appeared as globular structures on cellulosic film, as observed by AFM. Spreading a dense lignin layer on CN film gave rise to the disappearance of the InfraRed resonance bands related to the DHP aromatics. The film obtained from alternate layers of cellulose/DHP was transparent in visible light and had weak absorption in UV wavelengths. Optical properties measured in the visible wavelength range by ellipsometry and spectrophotometry indicated that beyond six bilayers (cellulose/DHP), the composite exhibits antireflexion properties.; L’affinité interfaciale entre une lignine modèle (dehydrogenopolymères [DHP]) et des nanocristaux de cellulose (CN) a été étudiée avant de construire un nanocomposite cellulose/DHP sous forme de multicouche par la méthode de spin-coating. L’isotherme d’adsorption de DHP, mesuré par ellipsométrie à l’interface liquide/film de CN, a montré que la concentration de surface de DHP adsorbés augmente avec la concentration du DHP en solution. Celui-ci a tendance à former des structures sous forme de globules observés par AFM sur le film cellulosique. Le dépôt d’une couche dense de lignine sur le film cellulosique se traduit par la disparition des bandes de résonance IR spécifiques des cycles aromatiques du DHP. Le film cellulose/DHP en couches alternées est transparent et absorbe légèrement dans l’UV. Au bout de six bicouches déposées, les propriétés optiques mesurées par ellipsométrie et spectrophotométrie montrent que le film possède des propriétés anti-réflexion.

International audience

The aim of this study was to understand how the molecular structures of amorphous polymers influence wood viscoelastic properties. Wood from oak and spruce was subjected to hydrothermal treatments at 110 or 135 degrees C. Wood rigidity, reflected by the wood storage modulus, showed different modification patterns according to the wood species or the temperature level. Because viscoelasticity is dependent on wood amorphous polymers, modifications of lignins and noncellulosic polysaccharides were examined. Hemicellulose degradation occurred only at 135 degrees C. In contrast, lignins displayed major structural alterations even at 110 degrees C. In oak lignins, the beta-O-4 bonds were extensively degraded and wood rigidity decreased dramatically during the first hours of treatment. Spruce lignins have a lower beta-O-4 content and, relative to oak, the wood rigidity decrease due to treatment was less pronounced. Wood rigidity was restored to its initial value by prolonged treatment, probably due to the formation of condensed bonds in cell wall polymers.

National audience