Combined hemicellulolytic and phenoloxidase activities of Thermobacillus xylanilyticus enable growth on lignin-rich substrates and the release of phenolic molecules.

Major challenge in biorefineries is the use of all lignocellulosic components, particularly lignins. In this study, Thermobacillus xylanilyliticus grew on kraft lignin, steam-exploded and native wheat straws produced different sets of phenoloxidases and xylanases, according to the substrate. After growth, limited lignin structural modifications, mainly accompanied by a decrease in phenolic acids was observed by Nuclear Magnetic Resonance spectroscopy. The depletion of p-coumaric acid, vanillin and p-hydroxybenzaldehyde combined to vanillin production in the culture media indicated that the bacterium can transform some phenolic compounds. Proteomic approaches allowed the identification of 29 to 33 different hemicellulases according to the substrates. Twenty oxidoreductases were differentially expressed between kraft lignin and steam-exploded wheat straw. These oxidoreductases may be involved in lignin and aromatic compound utilization and detoxification. This study highlights the potential value of Thermobacillus xylanilyticus and its enzymes in the simultaneous valorization of hemicellulose and phenolic compounds from lignocelluloses.

Dual Antioxidant Properties and Organic Radical Stabilization in Cellulose Nanocomposite Films Functionalized by In Situ Polymerization of Coniferyl Alcohol.

A new biobased material based on an original strategy using lignin model compounds as natural grafting additive on a nanocellulose surface through in situ polymerization of coniferyl alcohol by the Fenton reaction at two pH values was investigated. The structural and morphological properties of the materials at the nanoscale were characterized by a combination of analytical methods, including Fourier transform infrared spectroscopy, liquid chromatography combined with mass spectrometry, nuclear molecular resonance spectroscopy, electron paramagnetic resonance spectroscopy, water sorption capacity by dynamic vapor sorption, and atomic force microscopy (topography and indentation modulus measurements). Finally, the usage properties, such as antioxidant properties, were evaluated in solution and the nanostructured casted films by radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH•) scavenging tests. We demonstrate the structure-function relationships of these advanced CNC-lignin films and describe their dual functionalities and characteristics, namely, their antioxidant properties and the presence of persistent phenoxy radicals within the material.

Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment.

BACKGROUND: Biorefining of lignocellulosic biomass has become one of the most valuable alternatives for the production of multi-products such as biofuels. Pretreatment is a prerequisite to increase the enzymatic conversion of the recalcitrant lignocellulose. However, there is still considerable debate regarding the key features of biomass impacting the cellulase accessibility. In this study, we evaluate the structural and chemical features of three different representative biomasses (Miscanthus × giganteus, poplar and wheat straw), before and after steam explosion pretreatment at increasing severities, by monitoring chemical analysis, SEM, FTIR and 2D NMR. RESULTS: Regardless the biomass type, combined steam explosion pretreatment with dilute sulfuric acid impregnation resulted in significant improvement of the cellulose conversion. Chemical analyses revealed that the pretreatment selectively degraded the hemicellulosic fraction and associated cross-linking ferulic acids. As a result, the pretreated residues contained mostly cellulosic glucose and lignin. In addition, the pretreatment directly affected the cellulose crystallinity but these variations were dependent upon the biomass type. Important chemical modifications also occurred in lignin since the ß-O-4' aryl-ether linkages were found to be homolytically cleaved, followed by some recoupling/recondensation to ß-ß' and ß-5' linkages, regardless the biomass type. Finally, 2D NMR analysis of the whole biomass showed that the pretreatment preferentially degraded the syringyl-type lignin fractions in miscanthus and wheat straw while it was not affected in the pretreated poplar samples. CONCLUSIONS: Our findings provide an enhanced understanding of parameters impacting biomass recalcitrance, which can be easily generalized to both woody and non-woody biomass species. Results indeed suggest that the hemicellulose removal accompanied by the significant reduction in the cross-linking phenolic acids and the redistribution of lignin are strongly correlated with the enzymatic saccharification, by loosening the cell wall structure thus allowing easier cellulase accessibility. By contrast, we have shown that the changes in the syringyl/guaiacyl ratio and the cellulose crystallinity do not seem to be relevant factors in assessing the enzymatic digestibility. Some biomass type-dependent and easily measurable FTIR factors are highly correlated to saccharification.

Use of food and packaging model matrices to investigate the antioxidant properties of biorefinery grass lignins.

The antioxidant properties of grass lignins recovered from an alkaline industrial process and from different ethanol organosolv pretreatment processes were compared using two types of tests: (i) classical radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH(•)) scavenging tests in dioxane/water or ethanol and (ii) tests involving multiphasic systems (lipid dispersion in water or cellulose film suspended in ethanol). These multiphasic systems were representative of food and packaging matrices in view of high-value applications. All lignins, in solution or in the film, effectively scavenged radicals. Moreover, they were competitive with a food commercial rosemary extract to protect linoleic acid against oxidation. Whereas the DPPH(•) test in dioxane was not discriminant, differences appeared between lignins when the test was performed in ethanol or with the multiphasic systems. Moreover, radical scavenging activity was preserved in the film even after its immersion in ethanol. Structural analysis of lignins revealed that low-molar-mass phenolics, namely p-hydroxycinnamic acids and lignin depolymerization products, governed lignin antioxidant properties in the multiphasic systems.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Caffeoyl coenzyme A O-methyltransferase down-regulation is associated with modifications in lignin and cell-wall architecture in flax secondary xylem.

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT, EC 2.1.1.104) down-regulated-flax (Linum usitatissimum) plants were generated using an antisense strategy and functionally characterized. Chemical analyses (acetyl bromide and thioacidolysis) revealed that the lignin quantity was reduced and that the Syringyl/Guaïacyl (S/G) lignin monomer ratio was modified in the non-condensed lignin fraction of two independent down-regulated lines. These modifications were associated with altered xylem organization (both lines), reduced cell-wall thickness (one line) and the appearance of an irregular xylem (irx) phenotype (both lines). In addition UV microspectroscopy also indicated that CCoAOMT down-regulation induced changes in xylem cell-wall structure and the lignin fractions. Microscopic examination also suggested that CCoAOMT down-regulation could influence individual xylem cell size and identity. As a first step towards investigating the cellular mechanisms responsible for the unusual structure of flax lignin (G-rich, condensed), recombinant flax CCoAOMT protein was produced and its affinity for different potential substrates evaluated. Results indicated that the preferred substrate was caffeoyl coenzyme A, followed by 5-hydroxyconiferaldehyde suggesting that flax CCoAOMT possesses a small, but probably significant 5' methylating activity, in addition to a more usual 3' methylating activity.

Structure and chemical composition of bast fibers isolated from developing hemp stem.

Microscopic and chemical changes of hemp bast fibers were studied during the maturation from vegetative to grain maturity stages at both apical and basal regions of the stems. The content of protein was the main factor related to fiber maturation, whereas increased proportions of mannose and glucose and decreasing levels of galactose were also highly significant. Enhanced glucose deposition in apical fibers could be related to the gradual thickening of the fibers, whereas in basal regions the thickness of the fibers nearly reached the maximum at vegetative stages. In contrast, the extent of lignification remained close to 3-4% during plant growth. Hemp fiber lignins were rich in guaiacyl units and would be rather condensed in nature. In addition, the proportion of p-hydroxyphenyl units displayed a constant decline during maturation. A progressive chemical fractionation of hemp fibers provided further insights to the occurrence and nature of noncellulosic polysaccharides. Notably, these data pointed out that maturation is accompanied by a significant increase in water- and alkali-soluble components containing glucose- and mannose-related polymers and a decrease in arabinose and galactose components disrupted by diluted hydrochloric acid. Taken together, chemical features of the noncellulosic components suggest that the architecture of hemp fibers differs slightly from that of the more widely studied flax fibers.

Lignification in the flax stem: evidence for an unusual lignin in bast fibers.

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 Maüle reagents and KMnO(4)-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.

Structure, chemical composition, and xylanase degradation of external layers isolated from developing wheat grain.

The external layers of wheat grain were investigated during maturation with respect to chemical and structural features and xylanase degradability. Cytochemical changes were observed in the isolated peripheral tissues of the wheat grain at four defined stages following anthesis. Marked chemical changes were highlighted at 11 days after anthesis, for which protein and lipid contents varied weakly. The profile of esterified ferulic acid showed large variation in the maturing peripheral layers of grain in contrast to the deposition of ferulate dimers, p-coumaric and sinapic acids. Lignin was monitored at the latest stages of ripening, which corresponds to the cessation of reserve accumulation in the grain. Arabinoxylans (AX) reached a maximum at 20 days and did not display any significant change in arabinosyl substitution proportion until ripeness. When submitted to xylanase, all outer layers were similarly altered in the proportion of soluble AX except for the peripheral tissues of the 11-day-aged wheat grain that had very little AX. Aleurone and nucellar layers were mostly degraded, whereas pericarp stayed intact at all stages of maturation. This degradation pattern was connected with the preferential immunolocalization of xylanase in aleurone and nucellar layers irrespective of the developmental stages. Further chemical examination of the enzyme-digested peripheral tissues of the grain supports the facts that ferulic ester is not a limiting factor in enzyme efficiency. Arabinose branching, ferulic dimers, and ether-linked monomers that are deposited early in the external layers would have more relevance to the in situ degradability of AX.

Overexpression of a heterologous sam gene encoding S-adenosylmethionine synthetase in flax (Linum usitatissimum) cells: Consequences on methylation of lignin precursors and pectins.

The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens. This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells. The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins. Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells. Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls. The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.