Wood Esterification by Fatty Acids Using Trifluoroacetic Anhydride as an Impelling Agent and Its Application for the Synthesis of a New Bioplastic.

Fatty acids (FA) and their derivatives with long alkyl chain structures are good candidates for wood esterification to confer thermoplastic properties to wood. Nevertheless, they do not react easily with hydroxyl groups of wood. In this study, we investigated the reactivity of wood with various fatty acids of different chain lengths using trifluoroacetic anhydride (TFAA) as the impelling agent in various reaction conditions. Generally, the esterification of fatty acids without solvents resulted in higher Weight Percentage Gain (WPG) and ester content than the reaction in the presence of CH2Cl2. The esterification reaction could be performed effectively at room temperature, though an increased reaction temperature provoked degradation of the esterified wood. WPG of 67% was obtained for the C3 and 253% for the C16 alkyl chain analogs, respectively. Nevertheless, the ester content was fairly uniform, with values between 10.60 and 11.81 mmol ester/gram of wood for all chain lengths. A higher quantity of reagent led to higher ester content, which tended to stabilize after a ratio of 1:4 wood and TFAA/FA. The esterification reaction was performed rapidly, with an ester content between 7.65 and 9.94 mmol ester/gram of wood being achieved only after 15 min of reaction. Fourier transform infrared spectroscopy (FTIR) analysis was performed to confirm the drastic chemical changes of wood before and after esterification. Morphological observation by scanning electron microscope (SEM), softening measurement by thermomechanical analysis (TMA), and contact angle measurements demonstrated the possibility of esterified spruce wood being applied as a new bioplastic.

Use of Pyrolysis-Gas Chromatography/Mass Spectrometry as a Tool to Study the Natural Variation in Biopolymers in Different Tissues of Economically Important European Softwood Species.

Intraspecific macromolecule variation in stemwood, knotwood, and branchwood was studied using analytical pyrolysis with the intention of introducing a rapid working method to assess the variance in lignin content using analytical pyrolysis and highlight variability markers. The study was performed on Picea abies, Abies alba, and Pseudotsuga menziesii. Lignin determined via analytical pyrolysis-GC/MS (Py-lignin) can be used to identify variations in lignin content, compared to using classical Klason lignin values as a reference method for lignin determination, which requires a correction factor. Principal component analysis (PCA) was performed to identify biopolymer pyrolysis product markers for different species, tissues, or heights that could help highlight structural differences. Douglas fir was differentiated from spruce and silver fir in the levoglucosan amount. Guaiacol was more present in spruce wood, and creosol was more present in Douglas fir. Knotwood was structurally close to stemwood in spruce and silver fir, but there was a clear transition between stemwood and branchwood tissue in Douglas fir. Knotwood was differentiated by higher furan compounds. Branchwood was clearly separate from stemwood and knotwood and presented the same markers as compression wood in the form of phenylpropanoid lignins (H-lignin) as well as isoeugenol and vinyl guaiacol, the two most produced lignin pyrolysis products.

Knotwood and Branchwood Polyphenolic Extractives of Silver Fir, Spruce and Douglas Fir and Their Antioxidant, Antifungal and Antibacterial Properties.

The extractive contents of three softwood species largely used in the wood industry, namely Abies alba (Silver fir), Picea abies (spruce) and Pseudotsuga menziesii (Douglas fir), have been determined quantitatively for knots and at different points chosen along their branches, before analysis using high-performance liquid chromatography coupled with Mass Spectrometry (HPLC-MS). The results indicated that branchwood samples located in close proximity to the stem present high contents of extractives similar to those recorded for the knots. HPLC analysis showed quite similar chemical compositions, indicating that first cm of the branches could be considered as an additional source of knotwood. The antibacterial, antifungal and antioxidant activities of knot's extractives have been investigated with the dual objective of better understanding the role of high levels of secondary metabolites present in the knot and evaluating their potential for biorefinery applications. The antioxidant activity study showed that crude extracts of Douglas fir knotwood presented higher radical scavenging activity levels than the extracts of Silver fir and spruce, which presented more or less the same activities. Silver fir and spruce knotwood extracts presented higher antibacterial activity levels than the Douglas fir knotwood extracts did, while Douglas fir knotwood extracts presented more fungal growth inhibition than the spruce and fir knotwood extracts did. The structure-activity relationships indicate that radical scavenging and antifungal activities are associated with a higher relative quantity of flavonoids in the crude extracts, while higher relative quantities of lignans are associated with antibacterial activity.

Effect of Tannins Addition on Thermal Stability of Furfurylated Wood.

This article presents the effect of the addition of condensed tannins, used as a reticulation agent, on the polymerization of furfuryl alcohol during wood furfurylation, as well as the effect of these condensed tannins on the thermal stability of modified wood. Three kinds of dicarboxylic acids (adipic acid, succinic acid, and tartaric acid), as well as glyoxal, used as model of a wood reticulation agent, were used to catalyze the polymerization of furfuryl alcohol or tannin-furfuryl alcohol solutions. Impregnation of furfuryl alcohol or tannin-furfuryl alcohol solution into the wood, followed by curing at 103 °C for a specific duration, was performed for the wood modification. The thermal stability of the obtained tannin-furfuryl alcohol polymers and their corresponding modified woods was investigated. The leaching resistance and dimensional stability of the modified woods were also evaluated. Results indicated that the partial substitution of furfuryl alcohol by the tannins improved the polymerization reactivity in conditions where furfuryl alcohol alone did not lead to the formation of a solid polymeric material. The thermal stability and leaching resistance of the furfurylated wood in the presence of tannins were improved. Dimensional stability was also improved for furfurylated samples, but the effect of tannin addition was not so obvious, depending on the acidic catalyst used.

Fully wood based novel translucent and thermoplastic materials by solvent-free esterification.

Wood has been investigated for bioplastic production because of its abundance and biorenewability to reduce dependence on petro-based plastics. A series of experiments have been carried out to graft myristic acid, chosen as the fatty acid model, onto spruce sawdust using trifluoroacetic anhydride (TFAA) as the impelling agent without any solvent. The reaction was performed rapidly, leading to high ester content. Most of the hydroxyl groups in wood structure reacted with myristic acid, as demonstrated by FTIR and CPMAS 13C NMR. XRD measurements indicated a decrease in wood crystallinity. Myristic acid-esterified wood showed higher thermal stability by TGA and DSC and delivered several softening temperatures, as observed by TMA. Thermoplastic and translucent films were obtained after pressing at a high temperature. Scanning electron micrographs revealed that pressed esterified wood at the high temperature showed complete disappearance of fibrous structure to a smooth and homogenous surface, indicating that thermal fluidity was achieved during pressing. Esterified sawdust film also showed surface hydrophobicity by contact angle measurements.

Development of Water Repellent, Non-Friable Tannin-Furanic-Fatty Acids Biofoams.

Tannin-furanic foams were prepared with a good yield using the addition of relatively small proportions of a polyflavonoid tannin extract esterified with either palmitic acid, oleic acid, or lauric acid by its reaction with palmitoyl chloride, oleyl chloride, or lauryl chloride. FTIR analysis allowed us to ascertain the esterification of the tannin, and MALDI-TOF analysis allowed us to identify a number of multi-esterified flavonoid oligomers as well as some linked to residual carbohydrates related to the equally esterified tannin. These foams presented a markedly decreased surface friability or no friability at all, and at densities lower than the standard foam they were compared to. Equally, these experimental foams presented a much-improved water repellence, as indicated by their initial wetting angle, its small variation over time, and its stabilization at a high wetting angle value, while the wetting angle of the standard foam control went to zero very rapidly. This conclusion was supported by the calculation of the total surface energy of their surfaces as well as of their dispersive and polar components.

Glutathione Transferases: Surrogate Targets for Discovering Biologically Active Compounds.

Glutathione transferases comprise a large class of multifunctional enzymes, some involved in detoxification pathways. Since these enzymes are able to interact with potentially toxic molecules, they could be used as targets to screen for compounds with biological activity. To test this hypothesis, glutathione transferases (GSTs) from the white-rot fungus Trametes versicolor have been used to screen for antifungal molecules from a library of tropical wood extracts. The interactions between a set of six GSTs from the omega class and 116 extracts from 21 tropical species were quantified using a high-throughput thermal shift assay. A correlation between these interactions and the antifungal properties of the tested extracts was demonstrated. This approach has been extended to the fractionation of an Andira coriacea extract and led to the detection of maackiain and lapachol in this wood. Altogether, the present results supported the hypothesis that such detoxification enzymes could be used to detect biologically active molecules.

Yield and compositions of bark phenolic extractives from three commercially significant softwoods show intra- and inter-specific variation.

Tree bark is rich in commercially valuable secondary metabolites such as polyphenolic compounds like flavonoids and tannins. The yield and composition of bark extractives from Abies alba varies longitudinally within the stem. A. alba bark above the crown had the greatest extractive content, but the concentration of polyphenolic compounds was greatest below the crown. Here, we use a nonlinear model describing how bark extractive yields of A. alba, Picea abies and Pseudotsuga menziesii change with height, where differences among species are accounted for using different model coefficients. For all species there is longitudinal variability in extract yield. For P. abies and P. menziesii, the form of that variation differs depending on whether the bark was located at the same position in the stem as branches. Although the relationship form of total extractive yield differs between branched and un-branched samples, the relationship forms for individual compound yields does not change depending on branch presence. Despite trees from thinned stands having longer crowns and faster growth rates, indicative of greater photosynthetic activity, there was no evidence that thinning had affected either the yield or composition of extractives in these species. In P. abies, the proportions of flavonoids was higher in bark from the top of the tree, whereas epi-gallocatechin gallate was found in high proportions at the stem base. In P. menziesii bark extracts, taxifolin was the dominant compound, present in significantly higher proportions in bark towards the base of the stem.

A reverse chemical ecology approach to explore wood natural durability.

The natural durability of wood species, defined as their inherent resistance to wood-destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white-rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long-term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood-decaying fungi and also wood natural durability.

Molecular recognition of wood polyphenols by phase II detoxification enzymes of the white rot Trametes versicolor.

Wood decay fungi have complex detoxification systems that enable them to cope with secondary metabolites produced by plants. Although the number of genes encoding for glutathione transferases is especially expanded in lignolytic fungi, little is known about their target molecules. In this study, by combining biochemical, enzymatic and structural approaches, interactions between polyphenols and six glutathione transferases from the white-rot fungus Trametes versicolor have been demonstrated. Two isoforms, named TvGSTO3S and TvGSTO6S have been deeply studied at the structural level. Each isoform shows two distinct ligand-binding sites, a narrow L-site at the dimer interface and a peculiar deep hydrophobic H-site. In TvGSTO3S, the latter appears optimized for aromatic ligand binding such as hydroxybenzophenones. Affinity crystallography revealed that this H-site retains the flavonoid dihydrowogonin from a partially purified wild-cherry extract. Besides, TvGSTO6S binds two molecules of the flavonoid naringenin in the L-site. These data suggest that TvGSTO isoforms could interact with plant polyphenols released during wood degradation.

Derivatives of the Lignan 7'-Hydroxymatairesinol with Antioxidant Properties and Enhanced Lipophilicity.

The lignan 7'-hydroxymatairesinol (1), extracted from the knotwoods of fir (Abies alba), spruce (Picea abies), and Douglas fir (Pseudotsuga menziesii), exhibited unexpected reactivity when esterification reactions were attempted on the hydroxy group at position C-7'. To circumvent the rapid intramolecular cyclization procedure, leading quantitatively to the lignan conidendrin (7), a simple strategy for 7'-esterification of 1 under mild conditions (three steps, up to 80% overall yield) was developed. Compared to hydroxymatairesinol (1) (log K'w = 1.49), the derivatives (2-5) had increased lipophilicity with log K'w > 3.1, as determined by a UHPLC method. Compounds 1-5 exhibited potent antioxidant properties in the same range as the standards ascorbic acid and α-tocopherol (IC50 = 20-25 µM) and higher than that of BHT using a DPPH radical-scavenging assay.

Whole-cell biosensor of cellobiose and application to wood decay detection.

Fungal biodegradation of wood is one of the main threats regarding its use as a material. So far, the detection of this decaying process is empirically assessed by loss of mass, when the fungal attack is advanced and woody structure already damaged. Being able to detect fungal attack on wood in earlier steps is thus of special interest for the wood economy. In this aim, we designed here a new diagnostic tool for wood degradation detection based on the bacterial whole-cell biosensor technology. It was designed in diverting the soil bacteria Streptomyces CebR sensor system devoted to cellobiose detection, a cellulolytic degradation by-product emitted by lignolytic fungi since the onset of wood decaying process. The conserved regulation scheme of the CebR system among Streptomyces allowed constructing a molecular tool easily transferable in different strains or species and enabling the screen for optimal host strains for cellobiose detection. Assays are performed in microplates using one-day culture lysates. Diagnostic is performed within one hour by a spectrophotometric measuring of the cathecol deshydrogenase activity. The selected biosensor was able to detect specifically cellobiose at concentrations similar to those measured in decaying wood and in a spruce leachate attacked by a lignolytic fungus, indicating a high potential of applicability to detect ongoing wood decay process.

Hepatoprotective and antidiabetic effects of Pistacia lentiscus leaf and fruit extracts.

Pistacia lentiscus (Anacardiaceae) is commonly used in folk medicine to treat various diseases. The aim of the present study was to evaluate the hepatoprotective and antioxidant activities of extracts of P. lentiscus leaves (PL) and fruits (PF) against experimentally induced liver damage. Furthermore, characterization of extracts was attempted by a spectroscopic methodology (Fourier transform infrared spectroscopy) and high-performance liquid chromatography with diode array detection analysis. A hepatoprotective potential against paracetamol [165 mg/kg body weight (b.w.)] toxicity was noticed in mice pretreated with the same dose of PL or PF extract (125 mg/kg b.w.) or a combination of both (PL/PF 63/63 mg/kg b.w.), as revealed by an analysis of biochemical parameters (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities and total bilirubin). These results were confirmed by histological examination of the liver, which revealed significant protection against paracetamol-induced hepatic necrosis. Furthermore, PF extract exhibited a promising antidiabetic activity in streptozotocin-induced diabetic rats, similar to the reference drug glibenclamide (0.91 g/L), a result confirmed by in vitro inhibition of α-amylase. We demonstrated that the leaf crude extract showed the best effect in all tested methods, compared to its fruit counterpart, probably due to the presence of higher amounts of phenolic compounds, as determined by phytochemical and Fourier transform infrared spectroscopy analyses. Moreover, high-performance liquid chromatography with diode array detection led to the identification of six compounds for each part of the plant. Gallic acid, a characteristic compound of Pistacia species, was most abundant in leaves and fruits, while luteolin was detected for the first time in fruits. Obtained activities of P. lentiscus extracts may well be due, at least in part, to the presence of the above compounds.

The GSTome Reflects the Chemical Environment of White-Rot Fungi.

White-rot fungi possess the unique ability to degrade and mineralize all the different components of wood. In other respects, wood durability, among other factors, is due to the presence of extractives that are potential antimicrobial molecules. To cope with these molecules, wood decay fungi have developed a complex detoxification network including glutathione transferases (GST). The interactions between GSTs from two white-rot fungi, Trametes versicolor and Phanerochaete chrysosporium, and an environmental library of wood extracts have been studied. The results demonstrate that the specificity of these interactions is closely related to the chemical composition of the extracts in accordance with the tree species and their localization inside the wood (sapwood vs heartwood vs knotwood). These data suggest that the fungal GSTome could reflect the chemical environment encountered by these fungi during wood degradation and could be a way to study their adaptation to their way of life.

Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases.

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions.

Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including ß-etherase that specifically cleaves the ß-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular ß-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests.

Characterization of a Phanerochaete chrysosporium glutathione transferase reveals a novel structural and functional class with ligandin properties.

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional ß-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4' in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.

Unusual amount of (-)-mesquitol from the heartwood of Prosopis juliflora.

A large amount of flavonoid has been extracted and isolated from the heartwood of Prosopis juliflora, an exogenous wood species of Kenya. Structural and physicochemical elucidation based on FTIR, (1)H and (13)C NMR, GC-MS and HPLC analysis clearly demonstrated the presence of (-)-mesquitol as the sole compound without any noticeable impurities. The product was able to slow down oxidation of methyl linoleate induced by AIBN. The important amount and high purity of (-)-mesquitol present in the acetonic extract of P. juliflora could therefore be of valuable interest as a potential source of antioxidants from a renewable origin.

Evaluation of thermally modified Grevillea robusta heartwood as an alternative to shortage of wood resource in Kenya: Characterisation of physicochemical properties and improvement of bio-resistance.

Heat treatment of Grevillea robusta, a tropical wood species of low natural durability, was carried-out under inert conditions to improve its decay resistance. Resistance of heat treated samples was evaluated by malt agar block tests after three months of exposure to several wood rotting fungi. Also resistance of heat treated wood against termites was tested in the laboratory and in the field. Results showed that durability against fungi and termites was greatly improved after treatment. There was a good correlation between decay resistance and mass loss due to thermal treatment. Microscopic, FTIR and (13)C MAS NMR analysis were performed to characterize wood chemical and anatomical modifications that occur after treatment to understand the reasons of the durability improvement.