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Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography–mass spectrometry (GC–MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm−1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC–MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

International audience; Eucalyptus Kraft lignin was blended in various proportions with two different types of polyethylene (PE) (low-density polyethylene and post-consumer recycled [PCR]), with or without compatibilizer (PE-glycidyl methacrylate [GMA] or PE-2-hydroxyethyl methyl acrylate copolymers). Materials prepared by injection molding or extrusion casting were characterized for their thermal, antioxidant, antibacterial and mechanical properties. In addition, the photopermeability was assessed, as this property is specifically relevant to mulching applications. The use of compatibilizers enhanced the antioxidant properties which reached more than 3 h oxidation induction time when lignin was present at 20 wt% and beyond. Good antibacterial properties were obtained on Gram-positive bacteria when using PE-GMA as compatibilizer. The photopermeability of the materials was also reduced, reaching a transmittance lower than 5% throughout the whole measurement range (200-800 nm) in PCR. However, higher lignin contents led to a more brittle material and the overall processability of the material became more difficult, features that were not improved by compatibilizers. These materials are promising candidates for mulching film application, especially with PCR which still lacks high-volume market applications.

37 p.-6 fig.-2 tab.-1 graph. abst., In order to better understand bacterial depolymerization of lignin, a new analytical approach was proposed using Pseudomonas putida KT2440 as delignifying bacterium and Escherichia coli as non-delignifying control. Two different types of lignins, technical Kraft lignin and synthetic dehydrogenopolymer (DHP), were submitted to a bioconversion kinetic study over 7 days. The concomitant analysis of the supernatant acid-precipitable lignin fraction and water-soluble extractives by high-performance size-exclusion chromatography (HPSEC) and gas chromatography – mass spectrometry (GC–MS) highlighted the specific action of P. putida towards these substrates, with the transitory formation of phenolic metabolites (dihydroferulic acid for Kraft lignins and dimers for DHP) and the prevention of Kraft lignin self-assemblying. In both cases lignin apparent depolymerization followed by repolymerization was observed. The analysis of the bacterial pellets indicated the time-increasing content of lignins associated to bacterial cells, which could account for the apparent structural changes observed with E. coli in the supernatant., This work was supported by ADEME [N° 1401C0066] and by the European Commission [ERA-IB-14-055] in the framework of the LIGBIO project. The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). Research in TDHB's group was supported by BBSRC grant BB/M025772/1.

International audience; This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY)The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

A “waste-valorization” approach was developed to transform recalcitrant hydrolysis lignin (HL) from second-generationbioethanol productioninto multifunctional bio-based products. The hydrolysis lignin (HL) was extracted with aqueousacetone, yielding two fractions enriched in lignin and cellulose, respectively. The soluble hydrolysis lignin (SHL) was converted into anionic and cationic colloidal lignin particles (CLPs and c-CLPs). The insoluble cellulose-rich fraction was transformed intolignocellulosicnanofibrils that were further combined with CLPs or c-CLPs to obtainnanocomposite filmswithtailored mechanical properties,oxygen permeabilityand antioxidant properties. To enable prospective applications of lignin in nanocomposite films and beyond, CLPs and c-CLPs were also produced from a soda lignin (SL) and the influence of the lignin type on the particle size and ecotoxicity was evaluated. Finally, the carbon footprint of the entire process from hydrolysis lignin to films was assessed and an integration to industrial scale was considered to reduce the energy consumption. While most previous work utilizes purified lignin and pristine and often purified cellulose fibers to produce nanomaterials, this work provides aproof of conceptfor utilizing the recalcitrant lignin-rich side stream of thebioethanolprocess as raw material for functional nanomaterials and renewable composites.

International audience; The Zelcor book: an electronic knowledge book for the cascading use of biorefinery recalcitrant side streams into high added-value biobased products and fine chemicals The European Zelcor project (https://cordis.europa.eu/project/id/720303/fr) combines several scientific, technical competencies and know-how, as well as industrial expertises, for the valorisation of biorefineries by-products that are rich in lignins and humins. Considering the complexity and diversity of the scientific and technical fields relative to lignins and humins structure, to their extraction processes in biorefineries and to their valorisation into products, Zelcor has implemented a tool to organise the knowledge, for an efficient transmission and capitalisation within the consortium. A knowledge book was therefore constructed and delivered for this purpose. This hypermedia is specifically designed to facilitate the understanding of the biorefinery cascading strategy developed within Zelcor and of the major achievements. It is constituted by standardised graphical representations of concepts, validated by the Zelcor experts, and illustrating all achievements and results of the project.

openaire: EC/H2020/720303/EU//Zelcor Funding Information: This work was funded by the Bio Based Industry Joint Undertaking under the European Union's Horizon 2020 research and innovation programme within the Zelcor project (under the grant number No 720303 ), part of the COFILI project (grant number D201550245 ) for AFM measurements funded by the Grand Est Region and the European FEDER Programme and the Lignoxyl project for EPR measurements supported by the Agence Nationale de la Recherche (ANR) through the Carnot Institutes 3BCAR ( www.3bcar.fr ) and Qualiment ( https://qualiment.fr/ ) (no. 3 no. 19-CARN-001-01 and no. 16-CARN 001-01). The EPR data in this manuscript were obtained using equipment supported jointly by the French National Ministry of Research (PPF IRPE), the “Fondation pour la Recherche Médicale” (FRM DGE20061007745), and the CNRS (Department of Chemistry and Life Sciences). The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-552 0040-SPS). Funding Information: This work was funded by the Bio Based Industry Joint Undertaking under the European Union's Horizon 2020 research and innovation programme within the Zelcor project (under the grant number No 720303), part of the COFILI project (grant number D201550245) for AFM measurements funded by the Grand Est Region and the European FEDER Programme and the Lignoxyl project for EPR measurements supported by the Agence Nationale de la Recherche (ANR) through the Carnot Institutes 3BCAR (www.3bcar.fr) and Qualiment (https://qualiment.fr/) (no. 3 no. 19-CARN-001-01 and no. 16-CARN 001-01). The EPR data in this manuscript were obtained using equipment supported jointly by the French National Ministry of Research (PPF IRPE), the ?Fondation pour la Recherche M?dicale? (FRM DGE20061007745), and the CNRS (Department of Chemistry and Life Sciences). The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-552 0040-SPS). Publisher Copyright: © 2021 The Authors Copyright: Copyright2021 Elsevier B.V., All rights reserved. This study investigated the relationships between lignin molecular and supramolecular structures and their functional properties within cellulose-based solid matrix, used as a model biodegradable polymer carrier. Two types of derivatives corresponding to distinct structuration levels were prepared from a single technical lignin sample (PB1000): phenol-enriched oligomer fractions and colloidal nanoparticles (CLP). The raw lignin and its derivatives were formulated with cellulose nanocrystals or nanofibrils to prepare films by chemical oxidation or pressure-assisted filtration. The films were tested for their water and lignin retention capacities, radical scavenging capacity (RSC) and antimicrobial properties. A structural investigation was performed by infrared, electron paramagnetic resonance spectroscopy and microscopy. The composite morphology and performance were controlled by both the composition and structuration level of lignin. Phenol-enriched oligomers were the compounds most likely to interact with cellulose, leading to the smoothest film surface. Their RSC in film was 4- to 6-fold higher than that of the other samples. The organization in CLP led to the lowest RSC but showed capacity to trap and stabilize phenoxy radicals. All films were effective against S. aureus (gram negative) whatever the lignin structure. The results show the possibility to tune the performances of these composites by exploiting lignin multi-scale structure.

International audience; A high-throughput agarose gel electrophoresis (AGE) analytical method has been developed to separate lignin fractions according to their molecular weight (M w), charge, and shape. Operating conditions to effect separation of species have been evaluated along with imaging parameters. Kraft, soda (Protobind), and Organosolv lignins showed distinct differences in migration. Bands were cut, extracted, and cross-analyzed by gel permeation chromatography (GPC), 1 H NMR, and pyrolysis GC/MS to confirm their identity as lignin. The band intensity was correlated with lignin concentration by running serially diluted samples and imaging each lane to produce a precise calibration curve. The AGE technique was used to monitor and compare enzymatic, bacterial, chemical, and hydrothermal lignin digestions. Each method showed changes in lignin migration and band intensities over time. Low M w species were seen in samples collected from the anode buffer tank. Though requiring further development, the AGE method can provide structural information about the lignin and is accessible to biological and chemistry laboratories.

Even if the ocean represents a large part of Earth&rsquo, s surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.

BackgroundTermites account for natural biomass utilization systems (NBUS) that evolved the ability to overcome the overall recalcitrance of lignins towards lignocellulose transformation processes. With the objective of applying this capacity to the conversion of technical lignins produced by biorefineries, a higher wood-feeding termite species, Nasutitermes ephratae was fed with a commercial grass soda lignin (Protobind 1000, PB1000). The survival rates of Protobind 1000-fed termites were determined as well as changes in the structure of gut bacterial community and in the chemical composition of this technical lignin. ResultsThe ingestion of PB1000 by worker castes of N. ephratae was revealed by Pyrolysis-Gas Chromatography Mass Spectrometry (Py-GC/MS) analyses directly performed on termites. Survival rates were reduced by two –fold in the termites fed with PB1000 compared to controls. The relative abundance of Firmicutes and Bacteroidetes increased in the gut bacterial community of termites fed with PB1000. The digestion of PB1000 by termites triggered an increase in the syringyl-to-guaiacyl (S/G) ratios. These changes in the chemical composition of PB1000 in the gut of termites was marked by a decrease in relative content of free phenolic monomers.ConclusionThis work showed the abilities of digestive tract of a wood-feeding higher termite species, N. ephratae to metabolize the fraction of the volatile phenolic monomers of PB1000. Overall, our results provide insights into the bacterial lineage candidates for development of bacterial inoculum for pretreatment processes in valorization of technical lignin in biorefinery.

International audience

International audience; Within the Zelcor European project, INRA applied a newly-developed process in ionic liquids to depolymerise and functionalise lignin (increase the phenol content) to two reference lignin samples (Protobind 1000 from GreenValue and Futurol residue) and their fractions. Advantages of the process are to combine short reaction time (max 40 min) and low temperatures (max 110°C) compared to alternative catalytic processes. The process conditions were selected according to the prior optimisation performed on phenolic model compounds (monomer, dimer and milled wood isolated lignins ). Thioacidolysis, 31P NMR, and SEC were applied to the ethyl acetate soluble and insoluble reaction products so as to assess the degree of depolymerisation and demethylation. The results indicate that: i) a prior extraction of the ethyl acetate soluble compounds is beneficial to the process; ii) up to 30% solubility can be reached after the treatment of insoluble fractions; iii) demethylation occurs for all samples. The samples were then tested for antioxidant properties study to assess whether the process generates fractions with improved performances compared to the lignin samples and fractions already available. In parallel to the technical development of the process, security assessment has been undertaken through close interaction with INERIS to identify possible safety advantages of the process and to check that there is no major obstacle to future industrial development of the process.

International audience; Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-C alpha oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. O-18 labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-beta-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative P-31 NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to P-31 NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.

International audience; Two lignocellulosic agricultural residues, sunflower stalks and rape straw, were investigated as potential low-cost, non-food substrates for the production of triacylglycerols by the oleaginous, lignocellulolytic bacteria Streptomyces lividans. Chemical analysis of each type of residue revealed similar cell wall compositions in the polysaccharides and lignins of the two feedstocks, with high lignin beta-O-4 bond content compared to other angiosperms' lignin. Growing tests of Streptomyces lividans TK 24 were performed before and after sequential water and ethanol extraction by assessing bacterial fatty acid accumulation. All extracted and non-extracted samples were found to be substrates of the bacteria with fatty acid production ranging between 19% and 44% of the production obtained with arabinose as a reference substrate. The maximum conversion rate was obtained with the less lignified, non-extracted sample. This study suggests that lignocellulosic residues from oleaginous crops could be advantageously valorized by microbial bioconversion processes for the production of lipids of interest.

The present paper focuses on the effects of electron beam (EB) irradiation on thermoplastic materials based on destructurized starch including glycerol and water as plasticizers to assess the potentiality of cinnamyl alcohol as reactive additive capable of counterbalancing the degradation of the polysaccharide by inducing interchain covalent linkages. The tensile properties at break of test specimens of controlled composition submitted to EB irradiation at doses ranging from 50 to 200 kGy revealed the presence of competitive chain scission and bridging in samples containing cinnamyl alcohol at a relative concentration of 2.5% with regard to dry starch. The occurrence of crosslinking under particular conditions was evidenced by gel fraction measurements. The treatment under radiation was also applied to model blends including maltodextrin as a model for starch and the other ingredients to gain an insight into the radiation induced mechanisms at the molecular level. The presence of cinnamyl alcohol is found to limit degradation. Size exclusion chromatography and gel fraction allowed to monitor the effects and confirmed unambiguously the attachment of UV-absorbing chromophores onto the maltodextrin main chain. The combination of the obtained results demonstrates the possibility of altering in a favorable way the tensile properties of plasticized starch by applying high energy radiation to properly formulated blends including aromatic compounds like cinnamyl alcohol. (C) 2011 Elsevier Ltd. All rights reserved.

The reactivity and physicochemical properties of lignins are partly governed by their molar mass distribution. The development of reliable standard methods for determination of the molar mass distribution is not only relevant for designing technical lignins for specific applications, but also for monitoring and elucidating delignification and pulping processes. Size-exclusion chromatography (SEC) offers many advantages, such as wide availability, short analysis time, low sample demand, and determination of molar mass distribution over a wide range. A collaborative study has been undertaken within the “Eurolignin” European thematic network to standardise SEC analysis of technical lignins. The high-molar-mass fraction of polydisperse lignins was shown to be the main source of intra- and interlaboratory variations, depending on the gel type, elution solvent, detection mode, and calculation strategy. The reliability of two widespread systems have been tested: one based on alkali and a hydrophilic gel (e.g., TSK Toyopearl gel) and the other based on THF as solvent and polystyrene-based gels (e.g., Styragel). A set of practical recommendations has been deduced.

The objective of this study was to assess the influence of the peroxidase/coniferyl alcohol (CA) ratio on the dehydrogenation polymer (DHP) synthesis. The soluble and unsoluble fractions of horseradish peroxidase (HRP)-catalyzed CA dehydrogenation mixtures were recovered in various proportions, depending on the polymerization mode (Zutropf ZT/Zulauf ZL) and HRP/CA ratio (1.6-1100purpurogallin U mmol(-1)). The ZL mode yielded 0-57%/initial CA of unsoluble condensed DHPs (thioacidolysis yields

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.

Three alkali lignin samples (one from sugar cane bagasse and two from wheat straw), were compared in terms of structure and reactivity when incorporated within a starch matrix submitted to electron beam irradiation. Films with a 60/40 starch to lignin ratio as well as control films made from pure starch were prepared by a casting technique in an aqueous medium. With the objective to cross-link the polymers, films were treated with radiation doses of 200 and 400 kGy. The influence of casting and irradiation on the films was investigated at a molecular level using spectroscopic and chromatographic techniques. Infrared spectra of the native and irradiated films showed that the intensity of the structural modifications induced by the treatment was both dependent on the irradiation dose and the lignin sample. Two samples showing opposite behavior were selected for further lignin structural investigation and gas chromatography–mass spectrometry GC–MS determination of low-molecular-weight phenolics. These analyses provided some evidences that condensation/oligomerization reactions occurred when the more reactive system (starch/bagasse lignin) was subjected to electron beam treatment and that these reactions were probably mediated by ferulic acid. This study highlights the reactivity of alkali lignins and the potential of irradiations to increase starch/lignin compatibility.

23 ref.; International audience; The interfacial behavior of oleosins, the most abundant proteins from seeds oil bodies, was investigated using the pendant drop method at water/oil interfaces and compared to the behavior of beta-casein and lysozyme, proteins with contrasted emulsifying properties. Recombined high (rS3) and low (rS4) molecular weight oleosins comprising N-terminal histidine tags were purified to electrophoretic homogeneity. rS3 decreased the interfacial tension at the oil/water interface better than rS4, oleosins being more efficient than beta-casein. Oleosins formed aggregates when spread on noncompressed phospholipid (PL) films at the air/water interface as observed using a Langmuir-Blodgett balance equipped with a Brewster angle microscope. Oleosin spread at the surface of a compressed PL monolayer (5-20 mN/m) did not aggregate. Pressure increased immediately and proportionally to the amount of protein spread on the monolayer. The results stress the capacity of oleosins to be inserted in oil and in PL monolayers, which is of particular relevancy to their potential uses as water/oil emulsifiers.

International audience; The properties of monolayers of the dioxan lignin and of model dehydrogenation polymers spread at the air-water interface were investigated by the following techniques: surface pressure, surface potential, ellipticity, rheological analysis and molecular modeling. Information about the state of the monolayer was obtained from Surface pressure-area and surface potential-area isotherms. At small deposited quantities (1-2 mgm(-2)) a quasi-monolayer (2D) structure is formed. The values obtained for the mean area per monomer and for the mean dipole moment Lire coherent with previously reported data. as well as with the proposed Molecular model. At higher deposited quantities (3-6 mgm(-2)) a complex 3D network is built. These statements are supported by the results obtained by means of ellipsometry and surface rheology. Ellipsometry provides data for the stability of the 2D structure and for the slightly unstable 3D network. By using an original rheological approach. both structures Lire distinguished by their mechanical response when a dilatational stress is applied. The quasi-monolayer structure behaves as in elastic 2D medium during the compression, while two relaxation processes with characteristic time of 10 and 100 s were observed. The 3D network behaves as a Maxwell viscoelastic body during, the compression. while three relaxation processes with characteristic times of 1.10 and 100 s were detected.

The impact of ball milling on structural components of maize (Zea mays L.) stem was investigated in relation to enzymatic hydrolysis of carbohydrates. Ball milling of extractive-free maize stem material was carried out with different milling times up to 12 h. Carbohydrate conversion from ball-milled maize stem material with cellulolytic preparation Onozuka R-10 increased to 79% with increasing ball milling time up to 4 h, but did not thereafter increase further. Ball milling caused drastic depolymerization of hemicelluloses as revealed by increasing amounts of monosaccharides released in absence of enzymes. Infrared spectroscopy showed that cellulose crystallinity decreased within the first 6 h milling thereafter reaching a plateau. Thioacidolysis of solid residue fractions obtained after treatment of ball-milled samples with Onozuka R-10 suggested that no extensive degradation of lignin occurred during the milling. The effect of lignin structure on enzymatic hydrolysis of associated carbohydrates was further studied independently of cellulose crystallinity. Two lignin-carbohydrate fractions comprising hemicellulose and lignin with either high or low amount of aryl ether linkages were used as model materials. Lignins abundant in aryl ether linkages appeared more detrimental than condensed lignins to enzymatic hydrolysis of associated carbohydrates. (C) 2014 Elsevier B.V. All rights reserved.

Films were prepared from wheat starch and crude commercial lignosulfonates in presence of glycerol, either by thermal moulding or by casting. Moulded films are compared to standard plasticized starch films through their mechanical properties and their behavior with respect to water. Stress-strain mechanical tests show that incorporating 10% lignosulfonates modifies the tensile resistance of plasticized starchy films (ultimate stress decreases of a factor three and elongation at break increases of a factor two), independently of the composition of the lignosulfonates. Above 10%, distinct mechanical behaviors are observed between calcium and sodium lignosulfonate based films. Dynamic mechanical thermal analysis reveals a single glass transition temperature ranging from 16–40°C as a function of lignin content.

Lien vers la version éditeur: http://link.springer.com/article/10.1007/s10924-013-0570-6; International audience; Due to their polyphenolic structure lignins bear a number of interesting functional properties, such as antioxidant activity. Natural antioxidants are very much looked for in the aim of protection of light or oxygen sensitive goods and are being used in active packaging. Poly(lactide) (PLA)-lignin films were prepared by twin screw extrusion followed by thermo-compression using two different commercial sources of alkali lignins obtained from gramineous plants. A good dispersion of lignin in the matrix was observed. Mechanical properties of the compounded material were merely diminished and oxygen barrier properties slightly enhanced. The chromatographic study of the lignins revealed that the low molecular weight fraction of both lignins increased during the polymer processing. The migration of low molecular weight compounds in an ethanol/water solution simulating fatty foodstuff was performed and the antioxidant activity of the extract was analysed. It was found that the activity increases with increasing severity of the heat treatment because of the generation of free phenolic monomers during processing. These results open an interesting way for application of lignins as an active compound in packaging materials. Lignins do not impair the mechanical and barrier performance of the polymer and the plastics processing even allows for the generation of active substances.

Cette communication a été présentée à la Session 3 - Les phytosanitaires, l’eau, le sol, l’air : transferts et effets sur les écosystèmes, remédiation.; absent

Biorefinery of sugar cane is the first economic activity of Reunion Island. Some sugar cane manufactured products (juice, syrup, molasses) have antioxidant activities and are sources of both phenolic compounds and Maillard Reaction Products (MRP). The study aimed to highlight the global antioxidant activity of sugar cane stillage and understand its identity. Chromatographic fractionation on Sephadex LH-20 resin allowed the recovery of a MRP-rich fraction, responsible for 58 to 66% of the global antioxidant activity according to the nature of the sugar cane stillage (DPPH test), and a phenolic compounds-rich fraction for 37 to 59% of the activity. A good correlation was recorded between the antioxidant activity of the sugar cane stillage and its content in total reducing compounds amount (Folin-Ciocalteu assay), among them 2.8 to 3.9 g/L of phenolic compounds (in 5-caffeoylquinic acid equivalent). Preliminary experiments by HPLC-DAD-MS allowed to identify several free phenolic acids and gave clues to identify esters of quinic acids.

Sequential fractionation of extractive-free maize stems was carried out using two mild alkaline extractions (0.5 and 2 M NaOH, 20 degrees C, 24 h) before and after endoglucanase treatment. This procedure provided two lignin-carbohydrate fractions (LC1 and LC2) recovered after each alkali treatment. LC1 and LC2 contained 39% and 8% of the total lignin amount, respectively. These two fractions contained structurally distinct lignin molecules. While the content of resistant interunit bonds in lignin was 77% in LC1, it was increased up to 98% in LC2. Not unexpectedly, both alkali-soluble fractions contained substantial amount of p-coumaric and ferulic acids ether-linked to lignins. These results outline heterogeneity of maize stem lignins related to fractionation of grass materials. (C) 2013 Elsevier Ltd. All rights reserved.

The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis. (C) 2011 Elsevier Ltd. All rights reserved.

Six coniferyl alcohol-coniferaldehyde dehydrogenation copolymers (DHcoPs) were synthesized in order to determine the influence of an increased number of aldehyde functions on hydrolysis. After heterogeneous hydrolysis using acidic Montmorillonite K10 clay, the DHcoPs were thioacidolyzed and analyzed by gel permeation chromatography (GPC). Comparison of the thioacidolyzed products, with and without the hydrolysis step, showed that there was a greater proportion of condensation reaction in the absence of aldehyde. When the coniferaldehyde content in the initial synthetic mixture was more than 30% (w/w), only a low fraction of condensed products was generated during the K10 clay hydrolysis step. This suggests that condensation pathways are mainly due to the alcohol present in the γ-position in the DHcoPs. Investigation of the reactivity and the potential condensation of aldehyde and alcohol monomers under hydrolysis conditions showed the important conversion of coniferyl alcohol and conversely the stability of coniferaldehyde.

In this study, eight maize recombinant inbred lines were selected to assess both the impact of lignin structure and the impact of cell wall reticulation by p-hydroxycinnamic acids on cell wall degradability independently of the main "lignin content" factor. These recombinant lines and their parents were analyzed for in vitro degradability, cell wall residue content, esterified and etherified p-hydroxycinnamic acid content, and lignin content and structure. Lignin structure and esterified p-coumaric acid content showed significantly high correlation with in vitro degradability (r = -0.82 and r = -0.72, respectively). A multiple regression analysis showed that more than 80% of cell wall degradability variations within these 10 lines (eight recombinant inbred lines and their two parents) were explained by a regression model including two main explanatory factors: lignin content and estimated proportion of syringyl lignin units esterified by p-coumaric acid. This study revealed new biochemical parameters of interest to improve cell wall degradability and promote lignocellulose valorization.

The objective of this study was to adapt and improve an environmentally friendly and fast routine method for the analysis of ferulic and p-coumaric acids released from grass cell-walls by alkaline hydrolysis. This methodological development was performed on maize samples selected for their contrasted contents in ferulic and p-coumaric acids as a consequence of their different maturity stages (from stage of 7th leaf with visible ligule to stage of silage harvest). We demonstrate that the Carrez method is an efficient substitute to the common solvent-consuming extraction by ethyl acetate for the preparation of samples suitable for HPLC–ESI-MS analysis. We prove that it is possible to replace methanol by ethanol in the Carrez step and at last we propose a scale reduction of this procedure that offer a first step towards high throughput determinations. The new method leads to a solvent consumption reduced by a factor 100 and only requires ethanol as organic solvent.

The use of heterogeneous conditions involving Montmorillonite K10 clay was investigated as a mild alternative to homogeneous acidolysis of a lignin model. Guaiacyl Dehydrogenation Oligomers (DHOs) synthesized by horseradish peroxidase were selected as starting material. Hydrolysis products were analyzed by gel permeation chromatography and by HPLC/mass spectrometry. Several experimental parameters were studied such as catalyst and substrate concentration, as well as reaction solvent composition in order to minimize high molar mass product formation generated by recombination mechanisms. In both catalytic modes, the best hydrolysis conditions were similar in terms of solvent composition with dioxane/water (90/10, v/v) and catalyst H(+) concentration of about 0.01 mol/L. Although the homogeneous catalysis generated only 28% of low molecular weight (LMW) products (monomers and dimers), clay catalysis generated 35%. In light of the qualitative analysis, both catalytic methods gave the same products, thus supporting similar hydrolysis mechanisms.

In order to determine the mechanism of the earlier copolymerization steps of two main lignin precursors, sinapyl (S) alcohol and coniferyl (G) alcohol, microscale in vitro oxidations were carried out with a PRX34 Arabidopsis thaliana peroxidase in the presence of H(2)O(2). This plant peroxidase was found to have an in vitro polymerization activity similar to the commonly used horseradish peroxidase. The selected polymerization conditions lead to a bulk polymerization mechanism when G alcohol was the only phenolic substrate available. In the same conditions, the presence of S alcohol at a 50/50 S/G molar ratio turned this bulk mechanism into an endwise one. A kinetics monitoring (size-exclusion chromatography and liquid chromatography-mass spectrometry) of the different species formed during the first 24 h oxidation of the S/G mixture allowed sequencing the bondings responsible for oligomerization. Whereas G homodimers and GS heterodimers exhibit low reactivity, the SS pinoresinol structure act as a nucleating site of the polymerization through an endwise process. This study is particularly relevant to understand the impact of S units on lignin structure in plants and to identify the key step at which this structure is programmed.

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

This paper opens onto a general discussion on the development of new polymeric materials obtained from lignin blends. The aim is ( i ) to look for good polymer candidates to obtain a good compatibility with lignins (that is among semi polar polymers), and ( ii ) to look for good lignin candidates to obtain a good compatibility with polymers showing extreme behaviours (very polar, e.g. starch, or apolar, e.g. polypropylene). The compatibility is simply assessed through the blend morphology, as studied by visible microscopy. The morphology of the blends obtained from semi polar polymers is very sensitive to the variation of the solubility parameters. In a low range of polymer solubility parameters ( Δ δ = 1 cal cm − 3 ) , both heterogeneous and homogeneous systems are obtained. These blends could be easily improved by a careful choice in the polymer structure (particularly in the family of biodegradable polyesters); it could be possible also to take advantage of lignin variability to improve the compatibility. Only low molecular weight lignins are compatible with apolar and very polar matrixes . These compounds induce interesting specific properties, and original methods have to be looked for in order to improve their production. To cite this article: C. Pouteau et al., C. R. Biologies 327 (2004).

We investigated the potential of an electron-beam post-treatment to tailor the properties of 70/30 and 80/20 wt. extruded starch-lignin films. The effect of a 400 kGy radiation on films differing essentially by the kind of lignins incorporated (lignosulfonates/alkali lignins) was assessed both at the macroscopic and the molecular levels. Changes in the polymer molecular structure were studied by IR spectroscopy, by thioacidolysis as well as by model compound experiments. Electron beam-irradiation at 400 kGy, a rather high dose for processing natural polymers, alters to some extent the mechanical resistance of the starch-based materials. However this treatment substantially reduces the hydrophilic surface properties of the films, while not harming their biodegradability. Involved in radical cross-coupling reactions, lignin phenolic compounds are likely to play a primary role in the formation of a hydrophobic condensed network. This study suggests that lower irradiation doses might yield biomaterials with improved usage properties.

The increasing need for environmental protection together with the concerns over the future availability of petrochemical feedstock have led to the design and development of new degradable thermoplastic materials, based on renewable resources that are more friendly to the environment than the conventially used petroleum-based plastics (Chapman, 1994; Fritz et al., 1995; Chiellini and Solaro, 1996; Krochta and De Mulder-Johnston, 1996).

The structure and surface properties of spread layers of lignins at the air/water interface have been studied by neutron reflectivity, spectroscopic ellipsometry, and static and dynamic tensiometry. The layers hold 70% water, and the surface concentration of lignin measured by neutron reflectivity is less than the amount deposited. When the spread amount increases from 1 to 16 mg m-2, 65−12.5% of the deposited lignin is recovered at the interface. The absolute value of the Brewster ellipticity decreases slowly after spreading and tends toward quasi-equilibrium after 30 h, a fact which could be explained by a slow desorption from the interfacial layer to the bulk. After a compression of the interfacial layers, the surface pressure and the Brewster ellipticity exhibit a strong relaxation due, at least in part, to a desorption. However, the diffusion of lignin molecules from the layer to the substrate is limited because at equilibrium the neutron reflectivity and ellipsometry data show that a significant lay...

Films containing 0–30% of commercial kraft lignin mixed with wheat starch were obtained by extrusion followed by thermal moulding. The influence of lignin content on the film mechanical properties was evaluated by stress-strain tests run at two ambient relative humidity levels. At 58% relative humidity and up to 20% lignin, a very slight increase of elongation and stress at break was observed. At 71% relative humidity and up to 30% lignin, the film resistance to elongation significantly decreased, compared to the control starch film. Dissolution tests and water sorption isotherms showed that lignin actually reduces the overall water affinity of the films. These results are consistent with the hypothesis of a twophase material consisting of a hydrophilic starch matrix filled with hydrophobic lignin aggregates, as confirmed by microscopic observations. Casting experiments showed that the lower molecular weight fraction of kraft lignin may be responsible for a slight plastification of starch.

To elucidate the effect of lignin structure on the properties of lignin−starch composites, an industrial pine kraft lignin sample was fractionated by sequential extraction with organic solvents. Three fractions were obtained with different carbohydrate contents, infrared spectra, and molecular size distributions. In addition, an in-depth structural investigation was performed by GC−MS analyses of the low-molecular weight phenolics occurring in the native fractions or obtained by thioacidolysis. The properties of the lignin−starch composites made from pine kraft lignin or its fractions were evaluated, as compared to reference starch films. Although present in small quantities, the low-molecular weight fraction isolated from pine kraft lignin was observed to have a key role in the mechanical properties of the films, mainly as a starch plasticizer. Keywords: Lignin; starch; lignin utilization; lignin−starch composites

Two alkali-soluble polysaccharide fractions from the cell wall of Ulva ‘rigida’ were determined to be β-1,4-linked glucoxylans and a β–1,4-linked glucuronan by chemical and NMR spectroscopic analysis. The 13C NMR spectrum of water-soluble xyloglucuronorhamnan sulfate from Ulva ‘rigida’ referred to as ulvan is reported and the 13C and 1H NMR chemical shifts of its major repeating unit, the aldobiuronic acid β-D-GlcA-(1,4)-L-Rha, are given. The composition and gelling properties of ulvan from Ulva species from ‘green tides’ are also reported. The thermoreversible gel required both calcium and borate ions and the shear storage modulus G’ was ion concentration dependent. The mechanism of gelation and the associations of the different Ulva cell wall soluble polysaccharides are discussed.