Caroline Rémond

Effect of harvesting date on the composition and saccharification of Miscanthus x giganteus.

The chemical composition of the whole aerial biomass and isolated organs of Miscanthus x giganteus was examined for saccharification into fermentable sugars at early and late harvesting dates. Delayed harvest was mainly related to increased amounts of cell wall and ester-linked phenolic acids. Addition of an enzyme cocktail (cellulases, beta-glucosidase and xylanase) resulted in similar enzyme digestibilities at the two harvesting dates, ranging from 11-13% and 8-9% of the cellulose and arabinoxylan, respectively. However, the internodes, leaves and sheaths varied in cell wall content and composition and gave rise to different saccharification yields with internodes being the most recalcitrant organs. Non-cell wall fraction was estimated as the amount of material extracted by neutral detergent solution, and accounted for 23% of the whole aerial biomass harvested at an early date. However, saccharification yields from the miscanthus biomass did not change after soluble fraction removal. An ammonia pretreatment improved enzyme efficiency on early-harvested miscanthus, to a greater extent than on late-harvested biomass. This trend was confirmed for two different years of harvesting.

Enzymatic synthesis of alkyl β-D-xylosides and oligoxylosides from xylans and from hydrothermally pretreated wheat bran

Direct and efficient preparation of pentyl and octyl β-D-xylosides and oligoxylosides has been achieved from xylans and n-pentanol or n-octanol in aqueous medium with xylanases. The method has been successfully applied with xylooligosaccharides produced from hydrothermally pretreated wheat bran to produce octyl oligoxylosides. The pentose-based surfactants thus obtained exhibit good surface properties compared to other alkyl glycosides. These molecules represent interesting candidates for the production of new green surfactants.

The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth

BackgroundThermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization), were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth.ResultsOur results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran.ConclusionThis study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided resulted in dissimilar enzyme production profiles. These results indicate the importance of using different biomass sources to encourage the production of specific enzymes.

Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans.

Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery.

Saccharification of Miscanthus x giganteus , incorporation of lignocellulosic by-product in cementitious matrix

Given the non competition of miscanthus with food and animal feed, this lignocellulosic species has attracted attention as a possible biofuel resource. However, sustainability of ethanol production from lignocelluloses biomass would imply reduction in the consumption of chemicals and/or energetic means, but also valorization of the lignocellulosic by-product remaining from enzymatic saccharification. Introduction of these by-products into a cementitious matrix could be used in manufacturing a lightweight composite. Miscanthus biomass was submitted to chemical pretreatments followed by saccharification using an enzymatic cocktail. Residues from saccharification were then mixed with a cementitious matrix. Given their mechanical properties and a good adherence between cement and by-product, the hardened materials could be used. However, the delay in the beginning of setting time is too long, which prevents the direct use of by-product into cementitious matrix. Preliminary experiments using a setting accelerator in the cementitious matrix permitted significant reduction in the setting time delay.

β-Xylopyranosides: synthesis and applications

D-Xylose is the main constituent of hemicelluloses, the second most abundant polysaccharide in nature after cellulose. Thus, the β-xylopyranose motif is widely distributed in the plant kingdom. On the contrary, D-xylose is an unusual carbohydrate in mammalian cells, only present in the structures of proteoglycans, macromolecules with important biological functions. In recent years, β-xylopyranosides have attracted renewed interest due to the development of biomass-derived molecules and their numerous applications. In this review, we will first focus on general routes for the preparation of β-xylopyranosides according to the nature of the glycosidic linkage and the aglycone moiety by chemical and enzymatic pathways. The main uses of these molecules for different applications, such as activators in the biosynthesis of glycosaminoglycans, for enzyme inhibition and as surfactants will be presented.

Chemoenzymatic synthesis of “click” xylosides and xylobiosides from lignocellulosic biomass

Synthesis of bio-based molecules from plant biomass with chemoenzymatic pathways represents a challenging task for the development of green chemistry. In this context, an efficient two-step chemoenzymatic sequence has been achieved for the preparation of triazole-linked xylosides and xylobiosides from biomass-derived xylans. The synthesis of propargyl xyloside and xylobioside catalysed by a xylanase in an aqueous medium was first studied and improved according to different reaction parameters. Cycloaddition reactions between the terminal alkyne moiety of these xylosides or xylobiosides and various aliphatic, aromatic or functionalized azides (“click chemistry”) afforded various triazole-linked O-xylosides and O-xylobiosides in high yields. These molecules are of interest for different biological applications.

'Click'-xylosides as initiators of the biosynthesis of glycosaminoglycans: Comparison of mono-xylosides with xylobiosides.

Different mono‐xylosides and their corresponding xylobiosides obtained by a chemo‐enzymatic approach featuring various substituents attached to a triazole ring were probed as priming agents for glycosaminoglycan (GAG) biosynthesis in the xylosyltransferase‐deficient pgsA‐745 Chinese hamster ovary cell line. Xylosides containing a hydrophobic aglycone moiety were the most efficient priming agents. Mono‐xylosides induced higher GAG biosynthesis in comparison with their corresponding xylobiosides. The influence of the degree of polymerization of the carbohydrate part on the priming activity was investigated through different experiments. We demonstrated that in case of mono‐xylosides, the cellular uptake as well as the affinity and the catalytic efficiency of β‐1,4‐galactosyltransferase 7 were higher than for xylobiosides. Altogether, these results indicate that hydrophobicity of the aglycone and degree of polymerization of glycone moiety were critical factors for an optimal priming activity for GAG biosynthesis.

Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability.

This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.

Dynamic study of how the bacterial breakdown of plant cell walls allows the reconstitution of efficient hemicellulasic cocktails

Designing more efficient mixtures of enzymes is necessary to produce molecules of interest from biomass lignocellulosic fractionation. The present study aims to investigate the strategies used by the thermophilic and hemicellulolytic bacterium Thermobacillus xylanilyticus to fractionate wheat bran and wheat straw during its growth. Results demonstrated ratios and levels of hemicellulases produced varied during growth on both biomasses. Xylanase activity was mainly produced during stationary stages of growth whereas esterase and arabinosidase activities were detected earlier. This enzymatic profile is correlated with the expression pattern of genes encoding four hemicellulases (two xylanases, one arabinosidase and one esterase) produced by T. xylanilyticus during growth. Based on identification of the bacterial strategy, the synergistic efficiency of the four hemicellulases during the hydrolysis of both substrates was evaluated. The four hemicellulases worked together with high degree of synergy and released high amounts of xylose, arabinose and phenolic acids from wheat bran and wheat straw.