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La presente Tesis Doctoral aborda la caracterización química de las ligninas y los lípidos presentes en diversos materiales lignocelulósicos, incluyendo tanto residuos derivados de la actividad agrícola (cascarilla y paja de arroz, y podas de naranjo) como cultivos agrícolas (tallo de papiros). La caracterización detallada de la composición y estructura de las ligninas se llevó a cabo utilizando técnicas analíticas avanzadas como la Cromatografía de Permeación en Gel (GPC), la Pirólisis acoplada a Cromatografía de Gases y Espectrometría de Masas (Py-GC/MS), la espectroscopía de Resonancia Magnética Nuclear bidimensional (2D-RMN), y métodos de degradación química como la Derivatización Seguida de Rotura Reductora (DFRC). Por otro lado, la composición química de los compuestos lipofílicos se analizó mediante cromatografía de gases acoplada a espectrometría de masas (GC/MS). Los resultados obtenidos del análisis de ligninas muestran que las ligninas de la cascarilla y la paja de arroz, así como la de las podas de naranjo estaban enriquecidas en unidades guaiacilo (G), con cantidades menores de p-hidroxifenilo (H) y siringilo (S). En cambio, se observaron grandes diferencias en las ligninas del tallo de papiro; mientras que la lignina de la corteza estaba enriquecida en unidades G, la lignina de la médula estaba enriquecida en unidades S. Además, las ligninas de la cascarilla y de la paja de arroz así como las de la corteza y la médula del tallo del papiro presentaban importantes cantidades de p-cumaratos y ferulatos incorporados en sus estructuras. Las diferencias en la composición de las ligninas se reflejaron en el contenido de los enlaces entre las unidades de lignina, teniendo las ligninas enriquecidas en unidades G una mayor contenido de enlaces condensados. Especialmente relevante fue el descubrimiento de diversos flavonoides como la naringenina chalcona, y las flavanonas naringenina y dihidrotricina, incorporadas, junto a la tricina, al polímero de lignina de am

ydroxystilbenes are a class of polyphenolic compounds that behave as lignin monomers participating in radical coupling reactions during the lignification. Here, we report the synthesis and characterization of various artificial copolymers of monolignols and hydroxystilbenes, as well as low-molecular-mass compounds, to obtain the mechanistic insights into their incorporation into the lignin polymer. Integrating the hydroxystilbenes, resveratrol and piceatannol, into monolignol polymerization in vitro, using horseradish peroxidase to generate phenolic radicals, produced synthetic lignins [dehydrogenation polymers (DHPs)]. Copolymerization of hydroxystilbenes with monolignols, especially sinapyl alcohol, by in vitro peroxidases notably improved the reactivity of monolignols and resulted in substantial yields of synthetic lignin polymers. The resulting DHPs were analyzed using two-dimensional NMR and 19 synthesized model compounds to confirm the presence of hydroxystilbene structures in the lignin polymer. The cross-coupled DHPs confirmed both resveratrol and piceatannol as authentic monomers participating in the oxidative radical coupling reactions during polymerization.

Phenolic metabolism is rewired during internode development and in response to cinnamyl alcohol dehydrogenase perturbation in sorghum, with different sets of genes and accumulating compounds. The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Innovative schemes for lignocellulosic biomass refining could optimize the chemical use and biomass conversion efficiency, providing diversified biorefinery products. A new biorefinery scheme used mild alkaline-sulfite chemithermomechanical processing to pretreat sugarcane bagasse, producing highly digestible solids and diluted lignosulfonates in the black pretreatment liquor. Most of the glucan and xylan contained in the pretreated solids were converted into monosaccharides by saccharification with commercial enzymes. The undigested biomass solids remaining from the saccharification process were recycled with the existing alkaline-sulfite black pretreatment liquor. The optimized reaction conditions (159 degrees C and 13% NaOH amendment in the liquor) enhanced the lignosulfonate concentration from 7.1 +/- 0.3 to 33.5 +/- 0.7 g/L. Mass balance for the process indicates that most of the original sugarcane bagasse lignin were split into lignosulfonates contained in the resulfonation liquor (57.3%) and in the enzymatic hydrolysate (16.2%). 2D-NMR analysis of a lignosulfonate fraction recovered by ultrafiltration confirmed that approximately 9-10% of all beta-O-4' alkyl-aryl ether substructures were alpha-sulfonated. The combined chemical characteristics of the prepared lignosulfonate suggested its suitability for use in several lignosulfonate applications.

Papyrus, produced from the white pith of Cyperus papyrus L., has been used for millennia as the major writing support by ancient cultures, but there was no continuous papyrus production until modern times. Therefore, papyrus production had to be rediscovered. Modern Egyptian papyrus producers claim that their sheets possess 'the same physical and chemical properties as ancient papyri'. To study if this is accurate, commercially available papyrus sheets were compared to ancient papyri and papyri produced according to Pliny's historic description. Material characterization was performed with a focus on the potentially color-bearing lignin. Two-dimensional nuclear magnetic resonance spectroscopy, derivatization followed by reductive cleavage, and pyrolysis-gas chromatography/mass spectrometry were complemented with microscopy and tests for surface pH and sodium content. The lignin data in the native pith and commercial sheets were compared to 10 ancient samples from the Papyrus Museum Vienna. The analytical data clearly show the involvement of a strong alkaline treatment followed by chlorine bleaching for commercial papyri, as expressed by higher pH values, altered lignin structures, and chlorinated lignin compounds. The inclusion of an alkaline step in ancient papyrus manufacture is discussed but dismissed, since the alkali-treatment causes a huge decrease in lignin content, that was not found for the analysed ancient specimen. We assume that this additional treatment was introduced to obtain yellowish papyrus sheets meeting optical expectations of modern spectators. Linguistic and art historic evidence indicates that such a step would not have made sense in antiquity, since it was desired to produce white papyri.

We have examined a sustainable biorefinery process for the conversion of cotton gin trash (CGT), a waste from the cotton industry to multiple high value products. Herein, we found CGT native lignin to have unique features as it not only contains the p-hydroxyphenyl (H), guaiacol (G) and syringol (S) units, but structures of flavonoids, tyrosine, and phenylalanine associated with it. We have used the lignin-first approach to exploit its features in CGT and depolymerize it using argon gas and ethanol over Pd/C. The results show that 50% of the bio-aromatics is trans-isoeugenol, a precursor to produce vanillin. The cleavage of N-compounds from CGT during the fractionation process follows similar path as the rate of formation of the bio-aromatics. CGT impurities were shown to affect the reusability of the Pd/C catalyst. The fractionated cellulose-rich portion of CGT was converted to 39 mol% (80% purity) of 5-chloromethylfurfural (CMF), and a solid fuel which has a higher heating value of 20 MJ kg−1. The liquid extract, removed prior to CGT processing, was hydrogenated to produce C13–C17 liquid hydrocarbons, and the solid extract rich in nutrients is suitable for crops and plants growth. © 2022

Rice (Oryza sativa L.) straw is a highly abundant, widely available, and low cost agricultural waste that can be used as a source to extract valuable phytochemicals of industrial interest. Hence, in the present work, the chemical composition of the lipophilic compounds present in rice straw was thoroughly characterized by gas chromatography and mass spectrometry using medium-length high-temperature capillary columns, which allowed the identification of a wide range of lipophilic compounds, from low molecular weight fatty acids to high molecular weight sterols esters, sterol glucosides, or triglycerides in the same chromatogram. The most abundant lipophilic compounds in rice straw were fatty acids, which accounted for up to 6,400 mg/kg (41.0% of all identified compounds), followed by free sterols (1,600 mg/kg; 10.2%), sterol glucosides (1,380 mg/kg; 8.8%), fatty alcohols (1,150 mg/kg; 7.4%), and triglycerides (1,140 mg/kg; 7.3%), along with lower amounts of high molecular weight wax esters (900 mg/kg; 5.8%), steroid ketones (900 mg/kg; 5.8%), monoglycerides (600 mg/kg; 3.8%), alkanes (400 mg/kg; 2.6%), diglycerides (380 mg/kg; 2.4%), sterol esters (380 mg/kg; 2.4%), tocopherols (340 mg/kg; 2.2%), and steroid hydrocarbons (60 mg/kg; 0.4%). This information is of great use for the valorization of rice straw to obtain valuable lipophilic compounds of interest for the nutraceutical, pharmaceutical, cosmetic, and chemical industries. Moreover, this knowledge is also useful for other industrial uses of rice straw, as in pulp and papermaking, since some lipophilic compounds are at the origin of the so-called pitch deposits during pulping. Copyright © 2022 Rosado, Marques, Rencoret, Gutiérrez and del Río.

Lignin is largely produced by the oxidative radical coupling of the three monolignols, p-coumaryl, coniferyl, and sinapyl alcohols, which form the p-hydroxyphenyl, guaiacyl, and syringyl lignin units in the lignin polymer. However, it is increasingly appreciated that other “unconventional” phenolic compounds can also behave as lignin monomers participating in oxidative radical reactions during the lignification process and becoming integrated into the lignin polymer in some plants. Various phenolic compounds have been found incorporated into the lignin, including phenolic compounds derived from the monolignol biosynthetic pathway (e.g., monolignol ester conjugates, caffeyl and 5-hydroxyconiferyl alcohols, hydroxycinnamaldehydes, among others). More fascinating still was the discovery in some plants of a series of phenolic compounds arising from other polyphenolic biosynthetic pathways, including flavonoids (tricin, dihydrotricin, naringenin, naringenin chalcone), hydroxystilbenes (resveratrol, piceatannol, and isorhapontigenin, and their O-glucosylated counterparts piceid, astringin, and isorhapontin), and ferulic acid amides (feruloyltyramine, and diferuloylputrescine), that are integrated into the lignin polymer. All of these discoveries indicate that the lignin structure is far more complex than previously thought, that the definition of lignin should not be restricted to a polymer derived solely from the three traditional monolignols, and that other unconventional lignin monomers must be considered, thus expanding the established definition of lignin. The overriding factor remains, however, that lignification, the process of polymerization to the lignin polymer, allows this compositional flexibility because it is a simple chemical process. © 2022 Elsevier Ltd

Diferuloylputrescine has been found in a variety of plant species, and recent work has provided evidence of its covalent bonding into lignin. Results from nuclear magnetic resonance spectroscopy revealed the presence of bonding patterns consistent with homo-coupling of diferuloylputrescine and the possibility of cross-coupling with lignin. In the present work, density functional theory calculations have been applied to assess the energetics associated with radical coupling, rearomatization, and dehydrogenation for possible homo-coupled dimers of diferuloylputrescine and cross-coupled dimers of diferuloylputrescine and coniferyl alcohol. The values obtained for these reaction energetics are consistent with those reported for monolignols and other novel lignin monomers. As such, this study shows that there would be no thermodynamic impediment to the incorporation of diferuloylputrescine into the lignin polymer and its addition to the growing list of non-canonical lignin monomers.

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While CglUPO selectively produced "pure" epoxides (monoepoxides and/or diepoxides), MroUPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rHinUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of alpha-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.

Lignin is a phenylpropanoid polymer derived essentially from the oxidative radical coupling of three p-hydroxycinnamyl alcohols, the so-called monolignols: p-coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all originating from the shikimate-derived monolignol biosynthetic pathway, have been found to behave as lignin monomers in many plants, including the monolignol ester conjugates (with acetate, p-coumarate, ferulate, and p-hydroxybenzoate) or the incompletely methylated monomers caffeyl and 5-hydroxyconiferyl alcohols, among others. However, recent studies have revealed that some phenolic compounds derived from other biosynthetic pathways also act as true lignin monomers, participating in radical coupling reactions during lignification in several plants. This is the case for the flavone tricin, established as a monomer in the lignins from grasses and other monocots, and the newly discovered hydroxystilbenes (resveratrol, isorhapontigenin, and piceatannol) that are implicated as monomers in the lignification of palm fruit endocarps, or the stilbene glucosides that have been found incorporated into the lignin of Norway spruce bark. The discovery of ‘nonconventional’ phenolic precursors arising from other biosynthetic pathways than the canonical monolignol biosynthetic pathway expands the traditional definition of lignin and reveals that any phenolic compound that is delivered to the cell wall may be oxidized and incorporated into the lignin polymer during lignification, subject exclusively to its chemical compatibility.

Papyri belong to the oldest writing grounds in history. Their conservation is of the highest importance in preserving our cultural heritage, which is best achieved based on an extensive knowledge of the materials’ constituents to choose a tailored conservation approach. Thermogravimetric Analysis (TGA) has been widely employed to quantify cellulose and lignin in papyrus sheets, yielding reported lignin contents of 25% to 40%. In this work, the TGA method conventionally used for papyrus samples was repeated and compared to other lignin determination approaches (Klason-lignin and acetyl bromide-soluble lignin). TGA can lead to a large overestimation of the lignin content of commercial papyrus sheets (~27%) compared to the other methods (~5%). A similar overestimation of the lignin content was found for the pith and rind of the native papyrus plant. We concluded that the TGA method should, therefore, not be used for lignin quantification.

he monolignols, p-coumaryl, coniferyl, and sinapyl alcohol, arise from the general phenylpropanoid biosynthetic pathway. Increasingly, however, authentic lignin monomers derived from outside this process are being identified and found to be fully incorporated into the lignin polymer. Among them, hydroxystilbene glucosides, which are produced through a hybrid process that combines the phenylpropanoid and acetate/malonate pathways, have been experimentally detected in the bark lignin of Norway spruce (Picea abies). Several interunit linkages have been identified and proposed to occur through homo-coupling of the hydroxystilbene glucosides and their cross-coupling with coniferyl alcohol. In the current work, the thermodynamics of these coupling modes and subsequent rearomatization reactions have been evaluated by the application of density functional theory (DFT) calculations. The objective of this paper is to determine favorable coupling and cross-coupling modes to help explain the experimental observations and attempt to predict other favorable pathways that might be further elucidated via in vitro polymerization aided by synthetic models and detailed structural studies.

The transport sector is the second largest energy consumer in EU being responsible for 33% of the total energy consumption and for about 25% of the total greenhouse gas emissions, and relies on crude oils for 95% of its energy needs. Decarbonizing the transport sector to support climate and energy goals is recognized as a major challenge in the Energy Roadmap 2050). The development of biofuels from sustainable biomass feedstocks can play an important role in this regard, supporting fuel security and the EU objective of having at least 32% of transport fuels derived from renewable sources by 2030, according to the Renewable Energy Directive 2018/2001. Plant biomass is the main source of renewable materials in Earth and represents a potential source of renewable energy and biobased products. Biomass is available in high amounts at very low cost (as forest, agricultural or industrial lignocellulosic wastes and cultures) and could be a widely available and inexpensive source for energy, transportation (bio)fuels and (bio)products. Using biomass as energy source can lower EU’s external energy dependence and reduce greenhouse gas emissions. In this regard, R&I need to focus on the efficient production of advanced biofuels from renewable sources. This chapter covers the main challenges that need to be addressed in the near future for using biomass as a safe and clean source of energy. Different technologies are reviewed for the production of energy from biomass, including thermochemical, biochemical and chemical processing technologies. The biomass can be burned, transformed into a fuel gas through partial combustion, into a biogas through fermentation, into bioethanol through biochemical processes, into biodiesel, into a bio-oil or into a syngas from which chemicals and liquid fuels can be synthesized. Moreover, it is being increasingly recognized that the overall cost-effectiveness of biomass-to-biofuels pathways can increase significantly through co-production of higher

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in p-coumarate, leading to increased p-coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in‐depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size‐exclusion chromatography, 2D‐NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt‐stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D‐NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS‐based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3‐ and 4‐O‐feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

This perspective summarizes and compares computational results for the thermodynamics of bond dissociation, coupling, and rearomatization for a number of noncanonical lignin monomer–lignol combinations that have been found to occur experimentally. The noncanonical lignin monomers discussed are tricin, caffeyl alcohol, 5-hydroxyconiferyl alcohol, and piceatannol. Among dimeric combinations, the results for bond dissociation are generally similar, but in cases for which trimers have been reported (tricin-lignol adducts), this value can be quite variable, with stereochemical and structural preferences. Among the adducts examined thus far, the energies associated with quinone methide formation and rearomatization are not dissimilar and would not impede subsequent polymerization. These fundamental studies may help to elucidate how lignin monomers are incorporated into the lignin polymer, provide leads for targeted genetic modification, and be of use in deconstruction for the production of commodity chemicals.

Eucalyptus globulus (E. globulus) wood samples were treated in media containing acidified water and methyl isobutyl ketone (MIBK) in order to achieve the one-pot conversion of the hemicellulosic and cellulosic fractions into furfural and levulinic and formic and acetic acids, respectively. The reactions were performed under diverse operational conditions (180 or 190 °C, reaction times up to 70 min, liquid-to-solid ratios of 10 or 20 g aqueous phase/g wood, sulfuric acid concentrations of 0.5 or 1 g/100 g aqueous phase). Near-quantitative molar conversion of pentosans into furfural was achieved under conditions leading to limited generation of organic acids. In one-pot experiments performed under selected conditions, the overall yield of furfural and organic acids reached 45.2 g/100 g of wood. MIBK-soluble lignin was recovered from the organic phase by water precipitation at a yield of 17.4 g/100 g of wood, defining an effective method for the simultaneous delignification and polysaccharide conversion of E. globulus wood. The recovered lignin was characterized for structure (using pyrolysis-gas chromatography-mass spectrometry and heteronuclear single quantum coherence) and molar mass distribution. © 2020 American Chemical Society.

Lignins from different tree barks, including Norway spruce (Picea abies), eucalyptus (Eucalyptus globulus), mimosa (Acacia dealbata) and blackwood acacia (A. melanoxylon), are thoroughly characterized. The lignin from E. globulus bark is found to be enriched in syringyl (S) units, with lower amounts of guaiacyl (G) and p-hydroxyphenyl (H) units (H/G/S ratio of 1:26:73), which produces a lignin that is highly enriched in beta-ether linkages (83 %), whereas those from the two Acacia barks have similar compositions (H/G/S ratio of approximate to 5:50:45), with a predominance of beta-ethers (73-75 %) and lower amounts of condensed carbon-carbon linkages; the lignin from A. dealbata bark also includes some resorcinol-related compounds, that appear to be incorporated or intimately associated to the polymer. The lignin from P. abies bark is enriched in G units, with lower amounts of H units (H/G ratio of 14:86); this lignin is thus depleted in beta-O-4 ' alkyl-aryl ether linkages (44 %) and enriched in condensed linkages. Interestingly, this lignin contains large amounts of hydroxystilbene glucosides that seem to be integrally incorporated into the lignin structure. This study indicates that lignins from tree barks can be seen as an interesting source of valuable phenolic compounds. Moreover, this study is useful for tailoring conversion technologies for bark deconstruction and valorization.

Cereal brans constitute an unexploited source of valuable cell wall polysaccharides and phenolics. Sequential subcritical water extraction (SWE) of wheat, barley and rye bran enables the isolation of cell wall polysaccharides with targeted molecular structures. The extracts switched from glucan-rich to higher contents of feruloylated arabinoxylan (F-AX) with extraction time, resulting in significant radical scavenging activity. Structurally, wheat and rye F-AXs exhibited mostly single C(O)-3 arabinose substitutions, whereas barley bran F-AX showed a complex substitution pattern. The recalcitrance of the insoluble residues after SWE was revealed by the enrichment of F-AX populations with di-substituted arabinose units, and the presence of hydroxycinnamic acids, lignin and proteins. This study demonstrates the versatility of SWE to extract cell wall polysaccharides from cereal brans with tailored molecular structures and radical scavenging activity, with potential use in food and biomedical applications.

Lignin is conventionally defined as being formed by the oxidative polymerization of three main monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform as genuine lignin monomers in many plants, as is the case of the monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass.

As ever more component monomers are discovered, lignin can no longer be regarded as deriving from just the three canonical monolignols. Pathway intermediates and additional products of truncated biosynthesis are now established lignin monomers. The array of acylated monolignols continues to expand. Game-changing findings have demonstrated that phenolics from alternative pathways, including flavonoids and hydroxystilbenes, are also involved in lignification, expanding the traditional concept. Beyond the basic science intrigue, these findings propound exciting new avenues for valorizing lignins, or for producing more readily extractable or depolymerizable lignins, in crop and bioenergy plants.

In this study, we have found for the first time that various classes of flavonoids, namely naringenin chalcone, naringenin, dihydrotricin, and tricin, are incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. Identification of flavonoids was performed by analysis of the compounds released from the lignin by derivatization followed by reductive cleavage (the DFRC method), as well as by two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopic analysis, and comparing with authentic standards.

Lignin-like fractions isolated from several ancestral plants –including moss, lycophyte, horsetail, fern, cycad, and gnetophyte species– were structurally characterized by pyrolysis-GC/MS and 2D-NMR spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the moss and horsetail lignin samples, and the 2D-NMR experiments displayed intense correlations signals of guaiacyl (G) units in fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte and the gnetophyte also incorporated significant amounts of syringyl (S) units, characteristic of angiosperms, which appeared much later in plant evolution. Remarkably, the HSQC spectra also revealed the presence of flavonoid compounds never reported before in natural lignins.

Lignin is a phenylpropanoid polymer derived essentially from the oxidative radical coupling of three p-hydroxycinnamyl alcohols (so-called “monolignols”) differing in their degree of methoxylation, p-coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all deriving from the shikimate-derived monolignol biosynthetic pathway, have been found to behave as lignin monomers in many plants, including the monolignol ester conjugates (with acetate, p-coumarate, ferulate, and p-hydroxybenzoate) or the incompletely methylated monomers caffeyl and 5-hydroxyconiferyl alcohols, among others. However, recent studies have revealed that some phenolic compounds derived from other biosynthetic pathways also act as true lignin monomers participating in coupling and cross-coupling reactions during lignification in several plants. This is the case for the flavone tricin recently established as a monomer in the lignins from grasses and other monocots (and the flavanone naringenin found in the lignin in an fnsII rice mutant) or the newly discovered hydroxystilbenes (resveratrol, isorhapontigenin and piceatannol) that are implicated as monomers in the lignin of palm fruit endocarps. The discovery in lignins of ‘non-conventional’ phenolic precursors arising from other biosynthetic pathways expands the traditional definition of lignin, illustrates yet again the high metabolic plasticity of lignification, and reveals that any phenolic compound that is delivered to the cell wall may be oxidized and incorporated into the lignin polymer during lignification, subject exclusively to its chemical compatibility. On the other hand, these discoveries reveal that valuable flavonoids and hydroxystilbenes could also be obtained from agricultural resides (cereal straws or palm fruit shells) or from low-value lignin side-streams in lignocellulose processing mills, which will open new opportunities for the valorization of these residues that are considered as wastes.

Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports on the thermodynamics of the proposed reactions using density functional theory calculations. The results indicated that, in general, the energetics of both homo-coupling and cross-coupling are not dissimilar from those of the monolignol coupling, demonstrating the compatibility of piceatannol with the lignification process. Moreover, the DFT methods appear to predict the correct courses of post-coupling rearomatization reactions. © 2019

The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosum and Humicola insolens were capable of selectively hydroxylating isophorone to 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegerita was less regioselective, forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolens UPO full kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotula and Coprinopsis cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE software, and the distances from heme-bound oxygen in H2O2-activated enzyme to different substrate atoms, and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion of 10 mM isophorone was achieved with H. insolens UPO within nine hours, with total turnover numbers up to 5500. These biocatalysts, which only require H2O2 for activation, may represent a novel, simple and environmentally-friendly route for the production of isophorone derivatives.

Following an integrated approach, Eucalyptus nitens wood samples were subjected to consecutive stages of aqueous fractionation and organosolv delignification, in order to separate hemicelluloses (mainly converted into soluble products from the aqueous stage) from lignin (largely converted into soluble fragments in the organosolv stage) and from cellulose (accumulated in the solid phase from pulping). The compositions of selected reaction media were studied by selected spectrophotometric, spectrometric, chromatographic, and nuclear magnetic resonance methods; and the solid phases from treatments were studied by diffractometry and scanning electron microscopy. The experimental information from the above tasks provides a deep insight on the yields, properties and potential applications of the target fractions in the scope of biorefineries.

The impact of energy production by conventional technologies on the environment and human health has promoted transition policies towards a new model for the energy sector. In this context, it is essential to identify the key challenges which favour the global implementation of a clean, safe and efficient energy system, focused on the ways in which energy is produced and stored, and the management of existing resources and their emissions.

The present invention discloses a novel process for the chain shortening of carboxylic acids having a hydrocarbon chain, catalyzed by a peroxygenase, preferably by a fungal peroxygenase from Marasmius rotula ( Mro UPO), in the presence of a peroxide. The mechanism starts with an ±-oxidation generating an ±-hydroxy acid, which is further oxidized to a reactive ±-keto intermediate whose decarboxylation yields the one-carbon shorter acid. [EN], La invención se refiere a un proceso encimático para acortar la cadena de un ácido carboxílico con una cadena hidrocarbonada de al menos tres átomos de carbono. Más particularmente, la invención se refiere a un proceso para acortar la cadena en un carbono catalizado por una peroxigenasa fúngica.