Crystalline nanoxylan from hot water extracted wood xylan at multi-length scale: Molecular assembly from nanocluster hydrocolloids to submicron spheroids.

As a contribution to expand accessibility in the territory of bio-based nanomaterials, we demonstrate a novel material strategy to convert amorphous xylan preserved in wood biomass to hierarchical assemblies of crystalline nanoxylan on a multi-length scale. By reducing the end group in pressurized hot water extracted (PHWE) xylan to primary alcohol as a xylitol form with borohydride reduction, the endwise-peeling depolymerization is effectively impeded in the alkali-catalyzed hydrolytic cleavage of side substitutions in xylan. Nanoprecipitation by a gradual pH decrease resulted in a stable hydrocolloid dispersion in the form of worm-like nanoclusters assembled with primary crystallites, owing to the self-assembly of debranched xylan driven by strong intra- and inter-chain H-bonds. With evaporation-induced self-assembly, we can further construct the hydrocolloids as dry submicron spheroids of crystalline nanoxylan (CNX) with a high average elastic modulus of 47-83 GPa. Taking the advantage that the chain length and homogeneity of PHWE-xylan can be tailored, a structure-performance correlation was established between the structural order in CNX and the phosphorescent emission of this crystalline biopolymer. Rigid clusterization and high crystallinity that are constructed by strong intra- and inter-molecule interactions within the nanoxylan effectively restrict the molecular motion, thereby promoting the emission of ultralong organic phosphorescence.

Engineering a semi-interpenetrating constructed xylan-based hydrogel with superior compressive strength, resilience, and creep recovery abilities.

Recent advances in the area of hydrogel synthesis have been directed to enhance the mechanical properties and biocompatibility, which are critical in their use as functional biomaterials. In this work, a green and facile method is introduced to produce a hydrogel based on xylan, a plant-based heteropolysaccharide, that is shown to successfully form hydrogen-bonded, semi-interpenetrating polymer networks with polyvinyl alcohol. Upon crosslinking with sodium trimetaphosphate, the obtained hydrogels achieved an exceptional compressive strength (up to 84.2 MPa at a fracture strain of 90 %), which surpasses any polysaccharide-based hydrogels reported so far. The hydrogels were further shown to have high degradation temperature (350-370 °C), to be mechanically resilient with a form and creep recovery of 95 % (78 % stress after 1000 cycles under 30 % strain) and 98 % in height, respectively. All materials used in the preparation of the hydrogels were non-toxic and biocompatible, which makes the synthesized hydrogels suitable potential candidates for soft-tissue engineering and biomedical applications.

Localization of (+)-Catechin in Picea abies Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with Endoconidiophora polonica of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.

Lignans in Knotwood of Norway Spruce: Localisation with Soft X-ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-ray Spectroscopy.

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.

Structural and Thermal Analysis of Softwood Lignins from a Pressurized Hot Water Extraction Biorefinery Process and Modified Derivatives.

In this work we have analyzed the pine and spruce softwood lignin fraction recovered from a novel pressurized hot water extraction pilot process. The lignin structure was characterized using multiple NMR techniques and the thermal properties were analyzed using thermal gravimetric analysis. Acetylated and selectively methylated derivatives were prepared, and their structure and properties were analyzed and compared to the unmodified lignin. The lignin had relatively high molar weight and low PDI values and even less polydisperse fractions could be obtained by fractionation based on solubility in i-PrOH. Condensation, especially at the 5-position, was detected in this sulphur-free technical lignin, which had been enriched with carbon compared to the milled wood lignin (MWL) sample of the same wood chips. An increase in phenolic and carboxylic groups was also detected, which makes the lignin accessible to chemical modification. The lignin was determined to be thermally stable up to (273⁻302 °C) based on its Tdst 95% value. Due to the thermal stability, low polydispersity, and possibility to tailor its chemical properties by modification of its hydroxyl groups, possible application areas for the lignin could be in polymeric blends, composites or in resins.

Novel biorenewable composite of wood polysaccharide and polylactic acid for three dimensional printing.

Hemicelluloses, the second most abundant polysaccharide right after cellulose, are in practice still treated as a side-stream in biomass processing industries. In the present study, we report an approach to use a wood-derived and side-stream biopolymer, spruce wood hemicellulose (galactoglucomannan, GGM) to partially replace the synthetic PLA as feedstock material in 3D printing. A solvent blending approach was developed to ensure the even distribution of the formed binary biocomposites. The blends of hemicellulose and PLA with varied ratio up to 25% of hemicellulose were extruded into filaments by hot melt extrusion. 3D scaffold prototypes were successfully printed from the composite filaments by fused deposition modeling 3D printing. Combining with 3D printing technique, the biocompatible and biodegradable feature of spruce wood hemicellulose into the composite scaffolds would potentially boost this new composite material in various biomedical applications such as tissue engineering and drug-eluting scaffolds.

In Planta Localization of Stilbenes within Picea abies Phloem.

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.

Seasonal variation in formation, structure, and chemical properties of phloem in Picea abies as studied by novel microtechniques.

MAIN CONCLUSION: Phloem production and structural development were interlinked with seasonal variation in the primary and secondary metabolites of phloem. Novel microtechniques provided new perspectives on understanding phloem structure and chemistry. To gain new insights into phloem formation in Norway spruce (Picea abies), we monitored phloem cell production and seasonal variation in the primary and secondary metabolites of inner bark (non-structural carbohydrates and phenolic stilbene glucosides) during the 2012 growing season in southern and northern Finland. The structure of developing phloem was visualised in 3D by synchrotron X-ray microtomography. The chemical features of developing phloem tissues isolated by laser microdissection were analysed by chemical microanalysis. Within-year phloem formation was associated with seasonal changes in non-structural carbohydrates and phenolic extractive contents of inner bark. The onset of phloem cell production occurred in early and mid-May in southern and northern Finland, respectively. The maximal rate of phloem production and formation of a tangential band of axial phloem parenchyma occurred in mid-June, when total non-structural carbohydrates peaked (due to the high amount of starch). In contrast, soluble sugar content dropped during the most active growth period and increased in late summer and winter. The 3D visualisation showed that the new axial parenchyma clearly enlarged from June to August. Sub-cellular changes appeared to be associated with accumulation of stilbene glucosides and soluble sugars in the newest phloem. Stilbene glucosides also increased in inner bark during late summer and winter. Our findings may indicate that stilbene biosynthesis in older phloem predominantly occurs after the formation of the new band(s) of axial parenchyma. The complementary use of novel microtechniques provides new perspectives on the formation, structure, and chemistry of phloem.

Non-cellulosic heteropolysaccharides from sugarcane bagasse - sequential extraction with pressurized hot water and alkaline peroxide at different temperatures.

The xylan-rich hemicellulose components of sugarcane bagasse were sequentially extracted with pressurized hot-water extraction (PHWE) and alkaline peroxide. The hemicelluloses were found to contain mainly arabinoxylans with varying substitutions confirmed by different chemical and spectroscopic methods. The arabinoxylans obtained from PHWE were found to be more branched compared to those obtained after alkaline extraction. Sequential extraction could be useful for the isolation of hemicelluloses with different degree of branching, molar mass, and functional groups from sugarcane bagasse, which can be of high potential use for various industrial applications.

Cationised O-acetyl galactoglucomannans: synthesis and characterisation.

Water-soluble O-acetyl-galactoglucomannans (GGMs) can be obtained from Norway spruce by hot-water-extraction of the wood or as a side product by ultrafiltration of mechanical pulping waters. Cationic and amphiphilic polysaccharides and their derivatives are of interest for a number of applications and thus quaternary nitrogen moieties with cationic charge were grafted onto GGMs in the heterogeneous reaction to render a cationic polyelectrolyte. The degree of substitution was measured by elemental analysis of nitrogen, by quantitative (13)C NMR and interestingly also by polyelectrolyte titration and the results were congruent. NMR, matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI-TOF-MS), and FT-IR analysis were used to characterise the product. THF or DMSO with water enhanced the reaction efficiency and decreased M(w) reduction in comparison to plain water as a reaction media. Cationised GGM was also successfully acetylated. The cationic derivatives of hemicelluloses can potentially be utilised as polyelectrolyte layers in packaging and pharmaceutical applications.

Intensification of hemicellulose hot-water extraction from spruce wood in a batch extractor--effects of wood particle size.

The effect of five different wood particle size fractions between 0.5 and 12.5 mm on hot-water extraction of acetylated water-soluble hemicelluloses from spruce wood with a batch extraction setup at 170 °C was investigated. Extraction kinetics, with regard to particle size, was also studied. The purpose was to intensify the hemicellulose extraction for high molar mass hemicelluloses at high yield and purity. About 30% of the wood was dissolved and basically all the hemicelluloses could be extracted. The average molar masses of the extracted hemicelluloses decreased rapidly during the first 10 min of the extraction, but were not much affected by the difference in wood particle sizes. Smaller particles resulted in higher extraction rates. The reaction order was established to be of pseudo-first order for particles above 2mm and 1.5st order for particles smaller than 2mm. The effective diffusion coefficient was determined to be 9.11×10(-10) m(2)/s.

Separation of polymeric galactoglucomannans from hot-water extract of spruce wood.

Two methods for separation of polymeric galactoglucomannans (GGMs) from a hot-water extract of spruce wood, i.e., membrane filtration and precipitation in ethanol-water, were compared. Filtration through a series of membranes with different pore sizes separated GGMs of different molar masses, from polymers to oligomers. Only polysaccharides were precipitated in ethanol-water. With the optimal water content of 5-15%, the precipitated amount was about 6% on wood basis. The average molar mass of the precipitated polysaccharides was 10-12kDa with a molar mass range of 4-20kDa. GGMs comprised about 80% of the precipitated hemicelluloses. Other precipitated polysaccharides were mainly arabinoglucuronoxylans and pectins (rhamnogalacturonans). Analysis of a lignin-free, ethanol-precipitated GGM preparation by (13)C NMR spectroscopy verified that it was structurally almost identical with a GGM-rich ethanol precipitate obtained from spruce wood by extraction at much milder conditions, 90°C for 60min.

Hydrophobication and characterisation of O-acetyl-galactoglucomannan for papermaking and barrier applications.

Norway spruce O-acetyl-galactoglucomannans (GGM) are water-soluble hemicelluloses that have potential to be produced in large scale as a side product of the mechanical pulping industry or by hot-water extraction of wood. Chemical modification is often needed to tailor such water-soluble polysaccharides into industrially valuable compounds. In this work, treatment of GGM with butyric and benzoic anhydride in pyridine/dimethylformamide rendered GGM derivatives, which were hydrophobic and partially soluble in organic solvents. The degree of substitution can be adjusted by varying the quantity of the reagent and reaction temperature. The dn/dc value for the benzoyl ester of GGM was determined in 0.05 M LiBr DMSO in order to obtain accurate molar mass analysis with SEC-MALLS-RI. Novel substances with adjusted hydrophobicity can thus be prepared on one step synthesis from natural hemicelluloses, which then showed a slight increase in the molar mass upon esterification.

Effects of pH control with phthalate buffers on hot-water extraction of hemicelluloses from spruce wood.

Ground spruce wood was extracted with water at 170 °C at four different pH levels (3.8, 4.0, 4.2 and 4.4) achieved by using phthalate buffers. Static batch extractions were carried out in an accelerated solvent extractor (ASE-300). The extracted non-cellulosic carbohydrates, predominantly galactoglucomannans (GGMs), were characterised mainly by sugar unit analysis and molar mass determination. Compared to extraction with plain water, extractions with phthalate buffer solutions gave similar yields of non-cellulosic carbohydrates, but gave up to 70% less monosaccharides, and consequently higher molar masses of extracted GGMs. Moreover, at these pH levels, the hydrolysis of acetyl groups were decreased by 40% compared to extraction with plain water, thus maintaining the water solubility of GGMs. It is concluded that hot water extraction of hemicelluloses in high-molar-mass form (average Mw about 10 kDa) from wood in good yields (8% of wood) demands appropriate control of pH, to a level of about 4.

Kinetics of acid hydrolysis of water-soluble spruce O-acetyl galactoglucomannans.

Water-soluble O-acetyl galactoglucomannan (GGM) is a softwood-derived polysaccharide, which can be extracted on an industrial scale from wood or mechanical pulping waters and now is available in kilogram scale for research and development of value-added products. To develop applications of GGM, information is needed on its stability in acidic conditions. The kinetics of acid hydrolysis of GGM was studied at temperatures up to 90 degrees C in the pH range of 1-3. Molar mass and molar mass distribution were determined using size exclusion chromatography with multiangle laser light scattering and refractive index detection. The molar mass of GGM decreased considerably with treatment time at temperatures above 70 degrees C and pH below 2. The molar mass distribution broadened with hydrolysis time. A first-order kinetic model was found to match the acid hydrolysis. The reaction rate constants at various pH values and temperatures were calculated on the basis of the first-order kinetic model. Furthermore, the activation energy, E, was obtained from the Arrhenius plot. The activation energy E was 150 kJ mol (-1) for acid hydrolysis of spruce GGM. The apparent rate constant during acid hydrolysis increased by a factor of 10 with a decrease in pH by 1 unit, regardless of temperature. In addition, gas chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were applied to study the released GGM monomers and oligomers.