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BackgroundBiomass 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.ResultsThe 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.ConclusionsOur 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.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Background Lignocellulose biomass is known as a recalcitrant material towards enzymatic hydrolysis, increasing the process cost in biorefinery. In nature, filamentous fungi naturally degrade lignocellulose, using an arsenal of hydrolytic and oxidative enzymes. Assessment of enzyme hydrolysis efficiency generally relies on the yield of glucose for a given biomass. To better understand the markers governing recalcitrance to enzymatic degradation, there is a need to enlarge the set of parameters followed during deconstruction. Results Industrially-pretreated biomass feedstocks from wheat straw, miscanthus and poplar were sequentially hydrolysed following two steps. First, standard secretome from Trichoderma reesei was used to maximize cellulose hydrolysis, producing three recalcitrant lignin-enriched solid substrates. Then fungal secretomes from three basidiomycete saprotrophs (Laetisaria arvalis, Artolenzites elegans and Trametes ljubarskyi) displaying various hydrolytic and oxidative enzymatic profiles were applied to these recalcitrant substrates, and compared to the T. reesei secretome. As a result, most of the glucose was released after the first hydrolysis step. After the second hydrolysis step, half of the remaining glucose amount was released. Overall, glucose yield after the two sequential hydrolyses was more dependent on the biomass source than on the fungal secretomes enzymatic profile. Solid residues obtained after the two hydrolysis steps were characterized using complementary methodologies. Correlation analysis of several physico-chemical parameters showed that released glucose yield was negatively correlated with lignin content and cellulose crystallinity while positively correlated with xylose content and water sorption. Water sorption appears as a pivotal marker of the recalcitrance as it reflects chemical and structural properties of lignocellulosic biomass. Conclusions Fungal secretomes applied to highly recalcitrant biomass samples can further extend the release of the remaining glucose. The glucose yield can be correlated to chemical and physical markers, which appear to be independent from the biomass type and secretome. Overall, correlations between these markers reveal how nano-scale properties (polymer content and organization) influence macro-scale properties (particle size and water sorption). Further systematic assessment of these markers during enzymatic degradation will foster the development of novel cocktails to unlock the degradation of lignocellulose biomass.

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.

The distribution of lignin, 8-5′ and 8-8′ linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5′ linked lignin substructure), and no KM2 labeling (8-8′ linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5′ linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

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

Understanding the anaerobic deconstruction of recalcitrant lignocellulose remains challenging. Combining substrate composition and transcriptomic analyses, we shortlisted Ruminiclostridium cellulolyticum enzymes that modify lignocelullose and distinguished two members of the large SGNH hydrolase superfamily potentially enhancing lignocellulosic biomass degradation by acting on decorations of lignin and hemicelluloses but also on cross-links implicating lignin. Using genetic modifications, bioinformatics and biochemistry, we show they promote the plant cell wall ester-linked hydroxycinnamic acid derivatives release, a role never described for these proteins mainly synthesized by the restricted group of cellulolytic and cellulosome-producing bacteria. In addition to the recent observation of fungal limited lignin alterations in oxygen absence, this discovery is to the best of our knowledge, the first evidence of such anaerobic bacterial process that provides a better comprehension of the biogeochemical Earth’s carbon cycle. Furthermore, a better knowledge of the anaerobic plant biomass degradation could help to design non-fossil resources based biotechnological applications, a cornerstone of bioeconomy development. Ruminiclostridium cellulolyticum ’s lignocellulose anaerobic degradation involves two cellulosomal SGNH hydrolases targeting plant cell wall esterified hydroxycinnamic acids, a new function promoting the polysaccharides access via lignin unzipping.

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed ( L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of ( ), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase ( ) reporter gene, and the spatio-temporal regulation of gene expression in flaxseed was determined by histochemical and activity assays of . The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L.) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maule reagents and KMnO4-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl-syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical 'woody plant lignin', thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.

Biomass production and cell wall composition are differentially impacted by harvesting year and genotypes, influencing then cellulose conversion in miniaturized assay.Using a high-throughput miniaturized and semi-automated method for performing the pretreatment and saccharification steps at laboratory scale allows for the assessment of these factors on the biomass potential for producing bioethanol before moving to the industrial scale. The large genetic diversity of the perennial grass miscanthus makes it suitable for producing cellulosic ethanol in biorefineries. The saccharification potential and year variability of five genotypes belonging to × and were explored using a miniaturized and semi-automated method, allowing the application of a hot water treatment followed by an enzymatic hydrolysis. The studied genotypes highlighted distinct cellulose conversion yields due to their distinct cell wall compositions. An inter-year comparison revealed significant variations in the biomass productivity and cell wall compositions. Compared to the recalcitrant genotypes, more digestible genotypes contained higher amounts of hemicellulosic carbohydrates and lower amounts of cellulose and lignin. In contrast to hemicellulosic carbohydrates, the relationships analysis between the biomass traits and cellulose conversion clearly showed the same negative effect of cellulose and lignin on cellulose digestion. The miniaturized and semi-automated method we developed was usable at the laboratory scale and was reliable for mimicking the saccharification at the pilot scale using a steam explosion pretreatment and enzymatic hydrolysis. Therefore, this miniaturized method will allow the reliable screening of many genotypes for saccharification potential. These findings provide valuable information and tools for breeders to create genotypes combining high yield, suitable biomass composition, and high saccharification yields.