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Native lignin, a major structural polymer in lignocellulosic biomass, offers unique advantages as a feedstock for high-performance bio-based polymeric materials. In this work, depolymerized native ...

Pretreatments of lignocellulosic biomass prior to fast pyrolysis need to give a better yield and quality of pyrolysis oil and be practical to implement at a commercial scale. This study demonstrate...

Producing the next generation of thermoset polymers from renewable sources is an important sustainability goal. Hydrogenolysis of pinewood lignin was scaled up for the first time from lab scale to a 50 L pilot-scale reactor, producing a range of depolymerized lignin oils under different conditions. These lignin hydrogenolysis oils were glycidylated, blended with bisphenol A diglycidyl ether, and cured to give epoxy thermoset polymers. The thermal and mechanical properties of the epoxy polymers were assessed by differential scanning calorimetry, thermogravimetric analysis, flexural testing, and dynamic mechanical thermal analysis. Replacing up to 67% of the bisphenol A epoxy with the lignin oil epoxies resulted in cured epoxy polymers with improvements of up to 25% in flexural stiffness and strength. Considerable scope exists in simplifying and scaling up the hydrogenolysis process to produce depolymerized lignins that can substitute established petrochemicals in the quest for renewable high-performance thermoset polymers.

In situ catalytic fast pyrolysis of pretreated wood was investigated using a fluidized bed reactor. The pretreatments included acid-leaching, torrefaction, and a combined pretreatment of acid-leaching followed by torrefaction. Acid-leaching reduced amounts of biomass minerals introduced into the process. However, acid-leaching caused agglomeration during pyrolysis leading to reactor defluidization. Acid-leaching also resulted in an upgraded bio-oil that was less deoxygenated compared to the corresponding bio-oil obtained from raw wood. Conversely, torrefaction had a beneficial effect leading to an increased yield of upgraded bio-oil without affecting its chemical composition. Torrefaction of the acid-leached wood prevented agglomeration, reversed the negative effect acid-leaching had on bio-oil quality, and gave an improvement in upgraded bio-oil yield. These effects, combined with the removal of biomass minerals that are detrimental to the catalyst, suggests acid-leaching coupled with torrefaction is a p...

Acid leaching of lignocellulosic biomass prior to fast pyrolysis can increase the bio-oil yield and improve bio-oil quality. However, fast pyrolysis of acid-leached biomass in fluidized bed reactor...

Non-catalytic and catalytic pyrolysis of pretreated pine wood was conducted using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Biomass pretreatment included acid-leaching, torrefaction, and acid-leaching followed by torrefaction. Forty-five pyrolysis products were identified in the Py-GC/MS chromatograms and their peak area data evaluated by principal component analysis. In non-catalytic pyrolysis, acid-leaching pretreatment enhanced the levels of levoglucosan, while torrefaction pretreatment enhanced the proportion of catechols in the volatile products. Increasing both the temperature and catalyst loading in catalytic pyrolysis promoted the formation of aromatic hydrocarbons. At lower pyrolysis temperatures (450–500 °C), acid-leaching pretreatment resulted in slightly lower proportions of aromatics, while torrefaction pretreatment had the opposite effect. Overall, temperature and catalyst loading were considerably more important factors in catalytic fast pyrolysis of pine wood than biomass pretreatment as studied by Py-GC/MS.

Unlike a traditional review, this article does not summarise and discuss scientific progress in terms of traditional focus topics such as upstream processes or conversion and downstream processes, but instead provides examples proving that “Yes, we can make money from lignin and other bio-based resources”. The nine success stories from Scion's research are differentiated by the size of the product from designing trees with customised lignin and modifying wood dried by supercritical processing to bioaromatics from lignin hydrogenolysis, the scale of the product – from wood fibre plastic composites at commercial scale and lignin-rich bioadhesives to bio-based materials for 3D printing and the value of the product(s) – from lignin-based nanofibres and supercritical extraction of chemicals and compounds to utilising biomass side streams.

Pretreating lignocellulosic biomass with certain ionic liquids results in structural and chemical changes that make the biomass more digestible by enzymes. In this study, pine wood was pretreated with 1-ethyl-3-methylimidazolium chloride/acetate ([C2 mim]Cl and [C2 mim][OAc]) at different temperatures to investigate the relative importance of substrate features, such as accessible surface area, cellulose crystallinity, and lignin content, on enzymatic digestibility. The ionic liquid pretreatments resulted in glucan conversions ranging from 23% to 84% on saccharification of the substrates, with [C2 mim][OAc] being more effective than [C2 mim]Cl. The pretreatments resulted in no delignification of the wood, some loss of cellulose crystallinity under certain conditions, and varying levels of increased surface area. Enzymatic digestibility closely correlated with accessible surface area and porosity measurements obtained using Simons' staining and thermoporosimetry techniques. Increased accessible surface area was identified as the principal structural feature responsible for the improved enzymatic digestibility.

The synthesis of novel epoxy resins from lignin hydrogenolysis products is reported. Native lignin in pine wood was depolymerized by mild hydrogenolysis to give an oil product that was reacted with epichlorohydrin to give epoxy prepolymers. These were blended with bisphenol A diglycidyl ether or glycerol diglycidyl ether and cured with diethylenetriamine or isophorone diamine. The key novelty of this work lies in using the inherent properties of the native lignin in preparing new biobased epoxy resins. The lignin-derived epoxy prepolymers could be used to replace 25-75% of the bisphenol A diglycidyl ether equivalent, leading to increases of up to 52% in the flexural modulus and up to 38% in the flexural strength. Improvements in the flexural strength were attributed to the oligomeric products present in the lignin hydrogenolysis oil. These results indicate lignin hydrogenolysis products have potential as sustainable biobased polyols in the synthesis of high performance epoxy resins.

Mild hydrogenolysis has been compared with thioacidolysis as a method for degrading lignins in situ and in isolated form before analysis by gas chromatography/mass spectrometry and quantitative 31P nuclear magnetic resonance (NMR) spectroscopy. Both degradation methods gave similar levels of β-aryl ether-linked phenylpropane units that were released as monomers. Degradation by hydrogenolysis generally gave lower levels of total phenylpropane units when analyzed by 31P NMR, especially in the case of lignins with high levels of condensed units. Overall, these results indicate that mild hydrogenolysis could offer an alternative to thioacidolysis for probing lignin structure.

The Pd/C-catalysed hydrogenolysis of in-situ and isolated lignins from Pinus radiata wood was investigated to gain a more complete understanding of the factors affecting yield and composition of the hydrogenolysis products. Such hydrogenolysis products could potentially be refined into aromatic feedstock chemicals providing sustainable alternatives to petroleum-derived phenols. Lignins were converted into solvent-soluble oils composed of monomeric, dimeric and oligomeric products in high yields, up to 89% of the original lignin. The main monomer products were dihydroconiferyl alcohol and 4-n-propyl guaiacol. Dimeric and oligomeric compounds constituted 75% of the hydrogenolysis oils and were mainly composed of dihydroconiferyl alcohol and 4-n-propyl guaiacol units linked by β-5, 5-5, 4-O-5 and β-1 linkages. Hydrogenolysis of steam exploded wood gave lower yields of lignin hydrogenolysis products compared to unmodified wood due to fewer reactive aryl-ether linkages in the lignin.

A cDNA clone encoding the lignin-related enzyme caffeoyl CoA 3-O-methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P. radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p-hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide-soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT-deficient transgenic lines. Pyrolysis-GC/MS and 2D-NMR studies demonstrated that these reductions were due to depletion of G-type lignin. Correspondingly, the proportion of H-type lignin in PrCCoAOMT-deficient transgenic lines increased, resulting in up to a 10-fold increase in the H/G ratio relative to untransformed controls. 2D-NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho-diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D-NMR. Comparison of the 2D-NMR spectra from PrCCoAOMT-RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT-deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a “bonsai tree-like” appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated β-aryl ether (β-O-4 unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.

Pretreating lignocellulosic biomass with certain ionic liquids results in structural and chemical changes that make the biomass more digestible by enzymes. In this study, pine wood was pretreated with 1-ethyl-3-methylimidazolium chloride/acetate ([C2 mim]Cl and [C2 mim][OAc]) at different temperatures to investigate the relative importance of substrate features, such as accessible surface area, cellulose crystallinity, and lignin content, on enzymatic digestibility. The ionic liquid pretreatments resulted in glucan conversions ranging from 23% to 84% on saccharification of the substrates, with [C2 mim][OAc] being more effective than [C2 mim]Cl. The pretreatments resulted in no delignification of the wood, some loss of cellulose crystallinity under certain conditions, and varying levels of increased surface area. Enzymatic digestibility closely correlated with accessible surface area and porosity measurements obtained using Simons' staining and thermoporosimetry techniques. Increased accessible surface area was identified as the principal structural feature responsible for the improved enzymatic digestibility.

Nitrous acid deaminative depolymerisation was used to prepare three chitosan oligomer (CO) mixtures from high-molecular weight chitosan. These mixtures of chitosan oligosaccharides were analysed by electrospray ionisation mass spectroscopy, potentiometric titration and gel permeation chromatography. A method based on potentiometric titration of the amino groups of the oligomers gave an average degree of polymerisation (DP) for the three preparations of 5 (CODP5), 9 (CODP9) and 14 (CODP14). Chitosan acetate and the chitosan oligomer mixtures were assayed against Leptographium procerum, Sphaeropsis sapinea and Trichoderma harzianum on nutrient media. Leptographium procerum and S. sapinea growth was prevented by chitosan acetate and chitosan oligomers at concentrations of 0.3–0.4% (w/v), whereas T. harzianum was able to overcome the fungistatic action of these compounds. The oligomer preparation CODP14 exhibited superior specific activity to both CODP5 and chitosan acetate, suggesting an optimum molecular weight for bioactivity. All oligomer preparations were more effective at pH 4 than at pH 6. This result, in combination with the inactivity of N-acetylated CODP14, indicated that amino group protonation was an important factor for fungistatic activity. The CODP14 preparation was reduced with sodium borohydride and fractionated by alkali precipitation and ion exchange chromatography. Bioassays of these fractions pointed towards DP and degree of deacetylation (DD) as key factors in chito-oligosaccharide bioactivity. Conversely, the terminal aldehyde groups generated by depolymerisation did not contribute to the activity observed.

Direct acetylation-deisopropylation of methyl dehydroabietate 1 (R=CH3) occurs under Friedel-Crafts reaction conditions to give in 84% yield a 1:1 mixture of methyl 13-acetyl-podocarpa-8,11,13-trien-18-oate 2 and methyl 13-acetyl-10α-podocarpa-8,1,13-trien-18-oate 3 .

Various Pinus radiata wood components, solvent extracted and acetylated, were irradiated with ultraviolet light (λ max = 350nm). The XPS spectrum of the acetylated wood surface resembled that of cellulose acetate suggesting that acetylation removes lignin from the wood surface. The presence of acetylated lignin in the acetic anhydride solution following acetylation, identified by IR and NMR, supports this theory. This could be a major contributor to the bleaching of wood on acetylation. The XPS spectra of irradiated wood and lignin showed that photo-oxidation had occurred in both extracted wood, untreated lignin and acetylated samples. Demethoxylation and/or β-aryl ether cleavage in untreated irradiated lignin is supported by the XPS spectra while deacetylation of irradiated acetylated samples is indicated by decreased C 1 band intensity. Acetylation proved ineffective in preventing photo-oxidation of wooden surfaces The depletion of lignin on the wooden surface, (an artefact of acetylation) results in less light absorption thereby retarding photo-induced yellowing. Peroxide treatment of extracted radiata pine did not deplete surface lignin.

Suppression of the lignin-related gene cinnamoyl-CoA reductase (CCR) in the Pinus radiata tracheary element (TE) system impacted both the metabolite profile and the cell wall matrix in CCR-RNAi lines. UPLC-MS/MS-based metabolite profiling identified elevated levels of p-coumaroyl hexose, caffeic acid hexoside and ferulic acid hexoside in CCR-RNAi lines, indicating a redirection of metabolite flow within phenylpropanoid metabolism. Dilignols derived from coniferyl alcohol such as G(8-5)G, G(8-O-4)G and isodihydrodehydrodiconiferyl alcohol (IDDDC) were substantially depleted, providing evidence for CCR's involvement in coniferyl alcohol biosynthesis. Severe CCR suppression almost halved lignin content in TEs based on a depletion of both H-type and G-type lignin, providing evidence for CCR's involvement in the biosynthesis of both lignin types. 2D-NMR studies revealed minor changes in the H:G-ratio and consequently a largely unchanged interunit linkage distribution in the lignin polymer. However, unusual cell wall components including ferulate and unsaturated fatty acids were identified in TEs by thioacidolysis, pyrolysis-GC/MS and/or 2D-NMR in CCR-RNAi lines, providing new insights into the consequences of CCR suppression in pine. Interestingly, CCR suppression substantially promoted pyrolytic breakdown of cell wall polysaccharides, a phenotype most likely caused by the incorporation of acidic compounds into the cell wall matrix in CCR-RNAi lines.

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of β(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of β(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-β(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. β(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, β(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the β(1,4)-galactan backbone in pine. A β(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a β(1,4)-specific endogalactanase confirmed the production of β(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20°C, respectively. The β(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring β(1,4)-galactan in pine.

The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata , a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p -hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13 C- 1 H correlative NMR confirmed the increase in p -hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata .

The natural trait variation in Arabidopsis thaliana (L.) Heynh. accessions is an important resource for understanding many biological processes but it is underexploited for wood-related properties. Twelve A. thaliana accessions from diverse geographical locations were examined for variation in secondary growth, biomechanical properties, cell wall glycan content, cellulose microfibril angle (MFA) and flowering time. The effect of daylength was also examined. Secondary growth in rosette and inflorescence stems was observed in all accessions. Organised cellulose microfibrils in inflorescence stems were found in plants grown under long and short days. A substantial range of phenotypic variation was found in biochemical and wood-related biophysical characteristics, particularly for tensile strength, tensile stiffness, MFA and some cell wall components. The four monosaccharides galactose, arabinose, rhamnose and fucose strongly correlated with each other as well as with tensile strength and MFA, consistent with mutations in arabinogalactan protein and fucosyl- and xyloglucan galactosyl-transferase genes that result in decreases in strength. Conversely, these variables showed negative correlations with lignin content. Our data support the notion that large-scale natural variation studies of wood-related biomechanical and biochemical properties of inflorescence stems will be useful for the identification of novel genes important for wood formation and quality, and therefore biomaterial and renewable biofuel production.

Compression wood represents a unique challenge for biochemical processing of softwoods to biofuels and chemicals on account of its high lignin and galactan, and low glucan contents. Here we report the impact of ionic liquid pretreatment on the chemistry and enzymatic digestibility of Pinus radiata compression wood and opposite wood. Samples were pretreated using 1-ethyl-3-methylimidazolium acetate at 120 °C and 155 °C for 3 h. Hemicelluloses were preferentially extracted during the pretreatment and the recovered biomass was enriched in cellulose and lignin. The pretreatment caused structural modifications to the lignin and polysaccharides that included loss of ether linkages and formation of condensed structures in the lignin, reduced cellulose crystallinity and possible depolymerisation of polysaccharide chains. The enzymatic digestibility of the cellulose was significantly enhanced in both wood types after pretreatment. Approximately 90% of the glucan was converted to glucose after enzyme treatment for 24 h following pretreatment at 120 °C compared to only 3–8% in untreated samples. Because the ionic liquid pretreatment was equally effective on both compression wood and opposite wood the outcome of the saccharification was largely determined by the original chemical composition of the two wood types.