Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

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.

Influence of Surface Chemistry of Fiber and Lignocellulosic Materials on Adhesion Properties with Polybutylene Succinate at Nanoscale.

The production of bio-based composites with enhanced characteristics constitutes a strategic action to minimize the use of fossil fuel resources. The mechanical performances of these materials are related to the specific properties of their components, as well as to the quality of the interface between the matrix and the fibers. In a previous research study, it was shown that the polarity of the matrix played a key role in the mechanisms of fiber breakage during processing, as well as on the final properties of the composite. However, some key questions remained unanswered, and new investigations were necessary to improve the knowledge of the interactions between a lignocellulosic material and a polar matrix. In this work, for the first time, atomic force microscopy based on force spectroscopy measurements was carried out using functionalized tips to characterize the intermolecular interactions at the single molecule level, taking place between poly(butylene succinate) and four different plant fibers. The efficiency of the tip functionalization was checked out by scanning electron microscopy and energy-dispersive X-ray spectroscopy, whereas the fibers chemistry was characterized by Fourier-transform infrared spectroscopy. Larger interactions at the nanoscale level were found between the matrix and hypolignified fibers compared to lignified ones, as in control experiments on single lignocellulosic polymer films. These results could significantly aid in the design of the most appropriate composite composition depending on its final use.

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.

Atomic force microscopy reveals how relative humidity impacts the Young's modulus of lignocellulosic polymers and their adhesion with cellulose nanocrystals at the nanoscale.

Lignocellulosic biomass is receiving growing interest as a renewable source of biofuels, chemicals and materials. Lignocellulosic polymers and cellulose nanocrystals (CNCs) present high added-value potential in the nanocomposite field, but some issues have to be solved before large-scale applications. Among them, the interaction between polymers at the nanoscale and the effect of the external parameters on the mechanical properties have to be more precisely investigated. The present study aims at evaluating how the relative humidity affects the reduced Young's modulus of lignocellulosic films prepared with crystalline cellulose, glucomannan, xylan and lignin and how relative humidity changes their nanoscale adhesion properties with CNCs. Using atomic force microscopy and force volume experiments with CNC-functionalized levers, increasing the relative humidity is shown to decrease the Young's modulus values of the different films and promote their adhesion forces with CNCs. In particular, CNCs more strongly interact with glucomannan and lignin than xylan, and in the case of lignin, the oxidation of the film promotes strong variations in the adhesion force. Such results allow to better understand the lignocellulosic film properties at the nanoscale, which should lead to an improvement in the production of new highly added-value composites.

Enhancing the Antioxidant Activity of Technical Lignins by Combining Solvent Fractionation and Ionic-Liquid Treatment.

A grass soda technical lignin (PB1000) underwent a process combining solvent fractionation and treatment with an ionic liquid (IL), and a comprehensive investigation of the structural modifications was performed by using high-performance size-exclusion chromatography, 31 P NMR spectroscopy, thioacidolysis, and GC-MS. Three fractions with distinct reactivity were recovered from successive ethyl acetate (EA), butanone, and methanol extractions. In parallel, a fraction deprived of EA extractives was obtained. The samples were treated with methyl imidazolium bromide ([HMIM]Br) by using either conventional heating or microwave irradiation. The treatment allowed us to solubilize 28 % of the EA-insoluble fraction and yielded additional free phenols in all the fractions, as a consequence of depolymerization and demethylation. The gain of the combined process in terms of antioxidant properties was demonstrated through 2,2-diphenyl-1-picrylhydrazyl (DPPH. ) radical-scavenging tests. Integrating further IL safety-related data and environmental considerations, this study paves the way for the sustainable production of phenolic oligomers competing with commercial antioxidants.

Real Time and Quantitative Imaging of Lignocellulosic Films Hydrolysis by Atomic Force Microscopy Reveals Lignin Recalcitrance at Nanoscale.

Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed. It enables to accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.

Langmuir-Blodgett Procedure to Precisely Control the Coverage of Functionalized AFM Cantilevers for SMFS Measurements: Application with Cellulose Nanocrystals.

Atomic force microscopy (AFM) experiments with functionalized tips are currently one of the most powerful tools to locally measure adhesion forces via single-molecule force spectroscopy (SMFS) measurements. The main difficulty is to precisely control the attachment of biomolecules to the cantilever. Different chemistry procedures have been developed including the use of spacer molecules. Even if a process works well for small biomolecules such as antibodies, issues remain regarding nanoparticles or larger objects such as cellulose nanocrystals because it is difficult to precisely control their coverage and homogeneity. In this work, an original procedure based on the Langmuir-Blodgett (LB) technique was implemented for lever functionalization with cellulose nanocrystals and compared with classical chemical strategies. LB shows to be almost 6.0-fold more efficient than chemical procedure in terms of cellulose nanocrystals coverage attachment. Moreover, the LB technology provides advantage of not requiring linker molecules, which could have detrimental effects such as overestimation of the interaction force. The structural characterization and SMFS measurements of lignocellulosic polymers show that this strategy enables the precise control of the lever coverage, which improves the accuracy of the adhesion measurements. Such methodology is expected to strongly impact the AFM tip/tipless functionalization and SMFS measurements in different fields.

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type.

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.

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.

A zoom into the nanoscale texture of secondary cell walls.

BACKGROUND: Besides classical utilization of wood and paper, lignocellulosic biomass has become increasingly important with regard to biorefinery, biofuel production and novel biomaterials. For these new applications the macromolecular assembly of cell walls is of utmost importance and therefore further insights into the arrangement of the molecules on the nanolevel have to be gained. Cell wall recalcitrance against enzymatic degradation is one of the key issues, since an efficient degradation of lignocellulosic plant material is probably the most crucial step in plant conversion to energy. A limiting factor for in-depth analysis is that high resolution characterization techniques provide structural but hardly chemical information (e.g. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM)), while chemical characterization leads to a disassembly of the cell wall components or does not reach the required nanoscale resolution (Fourier Tranform Infrared Spectroscopy (FT-IR), Raman Spectroscopy). RESULTS: Here we use for the first time Scanning Near-Field Optical Microscopy (SNOM in reflection mode) on secondary plant cell walls and reveal a segmented circumferential nanostructure. This pattern in the 100 nm range was found in the secondary cell walls of a softwood (spruce), a hardwood (beech) and a grass (bamboo) and is thus concluded to be consistent among various plant species. As the nanostructural pattern is not visible in classical AFM height and phase images it is proven that the contrast is not due to changes in surfaces topography, but due to differences in the molecular structure. CONCLUSIONS: Comparative analysis of model substances of casted cellulose nanocrystals and spin coated lignin indicate, that the SNOM signal is clearly influenced by changes in lignin distribution or composition. Therefore and based on the known interaction of lignin and visible light (e.g. fluorescence and resonance effects), we assume the elucidated nanoscale structure to reflect variations in lignification within the secondary cell wall.

Modeling progression of fluorescent probes in bioinspired lignocellulosic assemblies.

Progression of enzymes in lignocellulosic biomass is a crucial parameter in biorefinery processes, and it appears to be one of the limiting factors in optimizing lignocellulose degradation. In order to assay the importance of the chemical and structural features of the substrate matrix on enzyme mobility, we have designed bioinspired model assemblies of secondary plant cell walls, which have been used to measure the mobility of fluorescent probes while modifying different parameters (probe size, water content, polysaccharide concentration). The results were used to construct a model of probe mobility and to rank the parameters in order of importance. Water content and probe size were shown to have the greatest effect. Although these assemblies are simplified templates of the plant cell walls, our strategy paves the way for proposing new approaches for optimizing biomass saccharification, such as selecting enzymes with suitable properties.

Natural organic UV-absorbent coatings based on cellulose and lignin: designed effects on spectroscopic properties.

Novel nanocomposite coatings composed of cellulose nanocrystals (CNCs) and lignin (either synthetic or fractionated from spruce and corn stalks) were prepared without chemical modification or functionalization (via covalent attachment) of one of the two biopolymers. The spectroscopic properties of these coatings were investigated by UV-visible spectrophotometry and spectroscopic ellipsometry. When using the appropriate weight ratio of CNC/lignin (R), these nanocomposite systems exhibited high-performance optical properties, high transmittance in the visible spectrum, and high blocking in the UV spectrum. Atomic force microscopy analysis demonstrated that these coatings were smooth and homogeneous, with visible dispersed lignin nodules in a cellulosic matrix. It was also demonstrated that the introduction of nanoparticles into the medium increases the weight ratio and the CNC-specific surface area, which allows better dispersion of the lignin molecules throughout the solid film. Consequently, the larger molecular expansion of these aromatic polymers on the surface of the cellulosic nanoparticles dislocates the π-π aromatic aggregates, which increases the extinction coefficient and decreases the transmittance in the UV region. These nanocomposite coatings were optically transparent at visible wavelengths.

Structure and optical properties of plant cell wall bio-inspired materials: cellulose-lignin multilayer nanocomposites.

Interfacial affinity between lignin model compound (dehydrogenation polymer [DHP]) and cellulose nanocristals (CN) was studied before building a nanocomposite cellulose/lignin in multilayer form by spin-coating method. The adsorption isotherm of DHP was measured by ellipsometry at the liquid/CN film interface and showed that the surface concentration of adsorbed DHP increases with the bulk concentration in solution. The DHP appeared as globular structures on cellulosic film, as observed by AFM. Spreading a dense lignin layer on CN film gave rise to the disappearance of the InfraRed resonance bands related to the DHP aromatics. The film obtained from alternate layers of cellulose/DHP was transparent in visible light and had weak absorption in UV wavelengths. Optical properties measured in the visible wavelength range by ellipsometry and spectrophotometry indicated that beyond six bilayers (cellulose/DHP), the composite exhibits antireflexion properties.

Elaboration of extensin-pectin thin film model of primary plant cell wall.

With the aim of mimicking the plant cell wall, a layer by layer approach was used to build a thin film consisting of successive adsorption of pectin and extensin. Elaboration of the thin film was monitored by surface plasmon resonance, quartz crystal microbalance, and ellipsometry. All data indicate that formation of the film was successful and that growth occurred according to a nonuniform growth. It is likely that diffusion of the polymers occurred within the multilayer structure and that the final structure is not constituted by layered individual pectin and extensin films. Polymer rearrangements were also supported by the atomic force microscopy images that show a smoother surface after extensin adsorption than after pectin deposition.

Langmuir-Blodgett films of cellulose nanocrystals: preparation and characterization.

The goal of this work is the preparation of monolayers of cellulose I nanocrystals providing flat crystalline cellulose surfaces. Suspensions of cellulose nanocrystals were prepared by hydrolyzing ramie and tunicin fibers with sulfuric acid. Due to surface grafted sulfate groups, the negatively charged, rod-like cellulose nanocrystals were found to form stable layers at the air-water interface in the presence of a cationic amphiphilic molecule such as dioctadecyldimethylammonium (DODA) used in this work. These layers were formed at different cellulose-DODA weight ratios, compressed and analyzed by tensiometry, ellipsometry and Brewster angle microscopy. At low cellulose concentrations the layers are discontinuous, becoming dense and homogeneous upon reaching a critical weight ratio, which depends on the aspect ratio of the cellulose nanocrystals. After transfer onto silicon wafers, the surface composition and morphology as well as the thickness of the films were examined by X-ray photoelectron spectroscopy, ellipsometry and atomic force microscopy. The results indicate that they are monolayer films, well structured, relatively smooth and pure. These films offer a crystalline and easily reproducible model cellulose surface.

Coniferyl alcohol reactivity at the air/water interface.

In order to investigate the sensitivity of the lignin monomer coupling reactions to the environment physicochemical conditions, coniferyl alcohol (CA) was polymerised at the air/water interface. Characterisation of the interface during the reaction by surface pressure measurement and ellipsometry demonstrates that the reaction occurs near or at the interface. Coupling products were analysed by HPLC and compared to reaction products obtained in the case of polymerisation in solution. Relative proportions of beta-beta and beta-O-4 dehydrodimers were found to increase in air/water interface experiment.

beta-casein and symmetrical triblock copolymer (PEO-PPO-PEO and PPO-PEO-PPO) surface properties at the air-water interface.

A comparison of beta-casein and symmetrical triblock copolymer (PEO-PPO-PEO and PPO-PEO-PPO) adsorption layer properties at the air-water interface has been carried out by bubble tensiometry and ellipsometry. It has been verified that the equation of state parameters (pi approximately gamma(y)) obtained from surface pressure (pi) and ellipticity in Brewster conditions (rhoB), which is proportional to the surface concentration (gamma) data, are the same as those obtained from dilational modulus epsilon and pi data. These two consistent approaches give further support to the theoretical model of block copolymers which has been previously developed for protein adsorption at fluid interfaces. It is shown that the interfacial behavior of the copolymer adsorption layer changes strongly as a function of the length of the hydrophilic and hydrophobic block sequences. The theoretical model may be used for the interpretation of the adsorption properties of the synthetic copolymers only when the size of the blocks is large enough. In the case of block copolymers, the coil is in a self-avoiding walk conformation (y = 3) whatever the temperature, while in the case of beta-casein, the polypeptide chain is partly collapsed at room temperature due to thermolabile noncovalent bonds. At the end of the first semidilute regime, there is clear evidence for a crossover toward a second semidilute regime for synthetic copolymers as well as for beta-casein but it is presently only partially characterized.