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One of the key challenges facing lignin conversion to fuels and chemicals is related to the level of carbohydrate and ash impurities found in extracted lignin. Structural modifications of lignin may also occur as a result of biomass pretreatment and harsh lignin extraction protocols. Extractive-Ammonia (EA) is a new pretreatment technology that uses liquid ammonia to cleave lignin–carbohydrate complexes, decrystallize cellulose, solubilize lignin, and selectively extract lignin from lignocellulosic biomass, enabling better utilization of both lignin and carbohydrate components in a biorefinery. The EA-based biorefinery produces two different lignin-rich streams, with different properties, that could potentially be upgraded to fuels and chemicals using green processes. In this work, a water/ethanol-based fractionation method was developed to enrich the ammonia-soluble extractives, resulting in a major product stream containing 92% lignin. Detailed characterization of the various streams resulting from EA treatment, including compositional analysis, structural characterization by nuclear magnetic resonance (NMR) spectrometry, elemental analysis, molecular weight analysis, and thermo-gravimetric analysis provides a broad evaluation of the EA-derived lignin product stream structures and properties, assessing their potential for commercial applications. In summary, EA-derived lignins preserve much of lignin's functionality, including the sensitive β-aryl ether units. Nitrogen incorporation was observed in the lignin-rich streams, notably due to the presence of hydroxycinnamoyl amides formed during ammonia pretreatment.

A new liquid ammonia pretreatment methodology called Extractive Ammonia (EA) was developed to simultaneously convert native crystalline cellulose Iβ (CI) to a highly digestible cellulose IIII (CIII) allomorph and selectively extract up to ∼45% of the lignin from lignocellulosic biomass with near-quantitative retention of all polysaccharides. EA pretreated corn stover yielded a higher fermentable sugar yield compared to the older Ammonia Fiber Expansion (AFEX) process while using 60% lower enzyme loading. The EA process preserves extracted lignin functionalities, offering the potential to co-produce lignin-derived fuels and chemicals in the biorefinery. The single-stage EA fractionation process achieves high biofuel yields (18.2 kg ethanol per 100 kg untreated corn stover, dry weight basis), comparable to those achieved using ionic liquid pretreatments. The EA process achieves these ethanol yields at industrially-relevant conditions using low enzyme loading (7.5 mg protein per g glucan) and high solids loading (8% glucan, w/v).

Various ferulic acid (FA) dimers occurring in plant cell walls, such as 8-5-, 8-O-4-, 5-5-, and 8-8-coupled dimers, are effective antioxidants and potential antimicrobials. It is necessary to access these diferulates as reference compounds to validate those isolated from plants. 3,6-bis(4-hydroxy-3-methoxyphenyl)-tetrahydrofuro-[3,4-c]furan-1,4-dione, a 8-8-coupled FA dilactone generated from ferulic acid via radical coupling, has been used to synthesize 8-8-coupled FA dimers although few reports investigated the distribution of products and mechanisms involved in the transformation of FA dilactone. In this work, the FA dilactone, obtained from FA by a peroxidase-catalyzed radical coupling, was reacted under various base/acid conditions. Effects of reaction conditions and workup procedures on the distribution of products were investigated by GC-MS. The isolated products from such treatments of FA dilactone were characterized by NMR. New derivatives of FA dimer including 2-(4-hydroxy-3-methoxybenzylidene)-3-(hydroxyl-(4-hydroxy-3-methoxyphenyl)methyl)succinic acid and 2-(bis(4-hydroxy-3-methoxyphenyl)-methyl)-succinic acid were produced from NaOH treatment. Another novel 8-8-coupled cyclic FA dimer, diethyl 6-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-methoxy-1,2-dihydronaphthalene-2,3-dicarboxylate was identified in products from FA dilactone treated by dry HCl in absolute ethanol. Mechanisms involved in such transformations were proposed.

&gamma, valerolactone (GVL)/H2O/acid solvent mixtures has been used in chemical pretreatment of lignocellulosic biomass, it was claimed that GVL lignins were structurally close to proto (native) lignins, or having low molecular weight with narrow polydispersity, however, the structural changes of GVL lignins have not been investigated. In this study, &beta, O-4 (&beta, aryl ether, GG), &beta, 5 (phenylcoumaran), and &beta, &beta, (resinol) lignin model compounds were treated by an acidic GVL-H2O solvent system, a promising pretreatment of lignocellulose for biomass utilization, to investigate the structural changes possibly related to the lignin involved. NMR characterization of the products isolated from the treated GG indicated that a phenyl dihydrobenzofuran, having typical C-H correlations at &delta, C/&delta, H 50.74/4.50 and 93.49/4.60 ppm in its HSQC spectrum, was produced from GG. In the pretreatment, the released formaldehyde from GG reacted fast with GG to form a novel 1,3-dioxane intermediate whose characteristic HSQC signals were: &delta, H 94.15&ndash, 94.48/4.81&ndash, 5.18 ppm and 80.82&ndash, 83.34/4.50&ndash, 4.94 ppm. The &beta, 5 model, dihydrodehydrodiconiferyl alcohol, was converted into phenylcoumarone and stilbene having benzaldehyde that resulted from the allyl alcohol side chain. The &beta, model, syringaresinol, was isomerized to form a mixture of syringaresinol, epi-, and dia-syringaresinol although being degraded slightly.

Chinese Angelica is a significant medical plant due to the various therapeutic constituents in its root, whereas the aerial part is considered worthless and often discarded as agricultural waste. In this work, phytochemicals from the stem were first systematically analyzed by means of GC&ndash, MS after derivatization and HPLC&ndash, MS/MS in multiple reaction monitoring (MRM) mode. Phthalides, ferulic acid, and coniferyl ferulate were detected in the stem, although their content is relatively low in comparison with the root. Some specific compounds, such as p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, caffeic acid, 4-hydroxyphenyl-1, 2-ethanediol, thymol-&beta, d-glucopyranoside, etc. and a significant amount of phytosterols (1.36 mg/g stem, mainly &beta, sitosterol) were detected in the stem. The extracted oil from the stem contained a considerable amount of phthalides (48.5 mg/g), &beta, sitosterol (56.21 mg/g), and stigmasterol (14.03 mg/g), no other bioactive compounds were found that could be potentially used as pharmaceuticals or additives to healthcare food.

Plant biologists are seeking new approaches for modifying lignin to improve the digestion and utilization of structural polysaccharides in crop cultivars for the production of biofuels, biochemicals, and livestock. To identify promising targets for lignin bioengineering, we artificially lignified maize (Zea mays L.) cell walls with normal monolignols plus 21 structurally diverse alternative monomers to assess their suitability for lignification and for improving fiber digestibility. Lignin formation and structure were assessed by mass balance, Klason lignin, acetyl bromide lignin, gel-state 2D-NMR and thioacidolysis procedures, and digestibility was evaluated with rumen microflora and from glucose production by fungal enzymes following mild acid or base pretreatments. Highly acidic or hydrophilic monomers proved unsuitable for lignin modification because they severely depressed cell wall lignification. By contrast, monomers designed to moderately alter hydrophobicity or introduce cleavable acetal, amide, or ester functionalities into the polymer often readily formed lignin, but most failed to improve digestibility, even after chemical pretreatment. Fortunately, several types of phenylpropanoid derivatives containing multiple ester-linked catechol or pyrogallol units were identified as desirable genetic engineering targets because they readily formed wall-bound polymers and improved digestibility, presumably by blocking cross-linking of lignin to structural polysaccharides and promoting lignin fragmentation during mild acidic and especially alkaline pretreatment.

In this study, 11 dehydrodiferulic acids (DFAs) and 8 diethyl dehydrodiferulates (DEFs) were synthesized and evaluated by Trolox equivalent antioxidant capacity (TEAC) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assays for their antioxidant properties to understand the Structure-Antioxidant Activity Relationship (SAR) of these dehydrodiferulates. In both assays, the order of antioxidant activity for all tested ferulic acid dimers were consistent except for 3-(4-Hydroxy-3-methoxy-benzylidene)-5-(4-hydroxy-3-methoxy-phenyl)-3H-furan-2-one (2, 8-8-lactone DC DFA, not occurred naturally) being the best antioxidant by TEAC test. The order of antioxidant activity of diferulic acid ethyl esters, evaluated by both assays, was not consistent; however, TEAC and DPPH assays provided consistent results for certain set of ethyl diferulates. In this study most of dimeric ferulates, with three exceptions, showed higher radical-scavenging efficacy than the monomers. Comparing the antioxidant activities of the tested diferulates suggested that the phenolic hydroxyl group, electron donating methoxyl group, and stable conjugated transient structures dictate the antioxidant activity of diferulates.

Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one] is a flavone that has been found to be incorporated in grass lignin polymers via 4′–O–β coupling. Herein, we investigated the tricin-lignin structure using nuclear magnetic resonance (NMR) methods by comparing the 1H–13C heteronuclear correlation (HSQC) NMR spectra of the isolated lignin with a series of dimeric and trimeric tricin-4′–O–β-ether model compounds. Results showed that the tricin moiety significantly affects the chemical shift of the Cβ/Hβ of 4′–O–β unit, producing peaks at around δC/δH 82.5–83.5/4.15–4.45, that differ from the Cβ/Hβ correlations from normal 4–O–β units formed solely by monolignols, and that have to date been unassigned., The authors thank the China Scholarship Council, State Education Department, for supporting living expenses for Wu Lan’s Program in the Department of Biological System Engineering, University of Wisconsin, Madison. W.L., F.Y., F.L., and J.R. (John Ralph) were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494 and DE-SC0018409). J.R. (Jorge Rencoret) and J.C.d.R. were funded by the Spanish Projects CTQ2014-60764-JIN and AGL2017-83036-R (co-financed by FEDER funds).

Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.

Recently, choline chloride/urea (ChCl/urea), a typical deep eutectic solvent (DES), has been found to possess various applications in organic synthesis, electrochemistry, and nanomaterial preparation. Herein we reported the first attempt to plasticize regenerated cellulose film (RCF) using ChCl/urea as an effective plasticizer. Meanwhile, RCFs plasticized with glycerol and sorbitol were also prepared for comparison. The plasticized RCFs were investigated by Fourier transform infrared (FT-IR) spectroscopy, wide-angle X-ray diffraction (XRD), atomic force microscopy (AFM), and mechanical testing. Transparent and soft RCFs could be successfully prepared in the presence of ChCl/urea, and high elongation at break (34.88%) suggested a significant plasticizing efficiency. No new crystal and phase separation occurred to ChCl/urea plasticized RCFs. The thermal stability of ChCl/urea plasticized RCF was lowered. These results indicated that ChCl/urea was an effective plasticizer for producing cellulose films.

22 páginas.-- 5 figuras.-- 2 tablas.-- 58 referencias.-- Supplemental Data inhttp://www.plantphysiol.org/content/167/4/1284/suppl/DC1, Open access.-- Artículo en acceso abierto, © 2014 American Society of Plant Biologists. All Rights Reserved. Tricinwas recentlydiscoveredinligninpreparations fromwheat (Triticum aestivum) strawandsubsequentlyinallmonocotsamplesexamined. To provide proof that tricin is involved in lignification and establish the mechanism bywhich it incorporates into the lignin polymer, the 4¢-O-b coupling products of tricinwith themonolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized alongwith the trimer that would result fromits 4¢-O-b-couplingwith sinapyl alcohol and then coniferyl alcohol. Tricinwas also found to cross couplewithmonolignols to form tricin-(4¢-O-b)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zeamays) lignin revealed that the tricinmoieties are found in even the highestmolecularweight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer.Thesefindingssuggest that tricin is fullycompatiblewithlignification reactions, isanauthentic ligninmonomer, and,because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers ofmonolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the lowmolecular mass compounds composed of flavonoid and lignan moieties., This work was supported by the Department of Energy Great Lakes Bioenergy Research Center (grant no. DE–FC02–07ER64494) and the Multidisciplinary Research Partnership Biotechnology for a Sustainable Economy (grant no. 01MRB510W) of Ghent University.

We demonstrate an experimental approach for upgrading lignin that has been isolated from corn stover via biomass fractionation using γ-valerolactone (GVL) as a solvent. This GVL-based approach can be used in parallel with lignin upgrading to produce soluble carbohydrates at high yields (≥70%) from biomass without the use of enzymes, ionic liquids, or concentrated acids. The lignin was isolated after an initial hydrolysis step in which corn stover was treated in a high-solids batch reactor at 393 K for 30 min in a solvent mixture consisting of 80 wt% GVL and 20 wt% water. Lignin was isolated by precipitation in water and characterized by 2D HSQC NMR, showing that the extracted lignin was similar to native lignin, which can be attributed to the low acid level and the low extraction temperatures that are achievable using GVL as a solvent. This lignin was upgraded using a two-stage hydrogenolysis process over a Ru/C catalyst. The isolated lignin was first dissolved to form a mixture of 10% lignin, 80% THF, 8.5% H3PO4 and 1.5% H2O, and treated at 423 K under hydrogen. The THF was removed by evaporation and replaced with heptane, forming a biphasic mixture. This mixture was then treated at 523 K in the presence of Ru/C and H2. The resulting heptane phase contained soluble lignin-derived monomers corresponding to 38% of the carbon in the original lignin. By adding 5% methanol during the second catalytic step, we produced additional monomers containing methyl esters and increased carbon yields to 48%. This increase in yield can be attributed to stabilization of carboxylic acid intermediates by esterification. The yield reported here is comparable to yields obtained with native lignin and is much higher than yields obtained with lignin isolated by other processes. These results suggest that GVL-based biomass fractionation could facilitate the integrated conversion of all three biomass fractions.

Underwater vibration detection is of great importance in personal safety, environmental protection, and military defense. Sealing layers are required in many underwater sensor architectures, leading to limited working‐life and reduced sensitivity. Here, a flexible, superhydrophobic, and conductive tungsten disulfide (WS2) nanosheets‐wrapped sponge (SCWS) is reported for the high‐sensitivity detection of tiny vibration from the water surfaces and from the grounds. When the SCWS is immersed in water, a continuous layer of bubbles forms on its surfaces, providing the sensor with two special abilities. One is sealing‐free feature due to the intrinsic water‐repellent property of SCWS. The other is functioning as a vibration‐sensitive medium to convert mechanical energy into electric signals through susceptible physical deformation of bubbles. Therefore, the SCWS can be used to precisely detect tiny vibration of water waves, and even sense those caused by human footsteps, demonstrating wide applications of this amphibious (water/ground) vibration sensor. Results of this study can initiate the exploration of superhydrophobic materials with elastic and conductive properties for underwater flexible electronic applications.

Bacterial cellulose (BC) is a green biopolymer suitable for heavy metal ion removal from aqueous solution due to its nano-porous microstructure. Polyethyleneimine-bacterial cellulose (PEI-BC) was prepared by reductive amination of dialdehyde BC with polyethyleneimine. The capacity of PEI-BC in Cu(II) and Pb(II) adsorption from aqueous solution was investigated. The adsorption kinetics could be well expressed by pseudo-second-order model and the adsorption isotherm data were well fitted with Freundlich model. Adsorption processes of Cu(II) and Pb(II) by PEI-BC reached equilibrium very rapid in 30 and 60min, respectively. The maximum adsorption capacity of PEI-BC on Cu(II) and Pb(II) was found to be 148 and 141mg/g, respectively, which was higher than that of unmodified BC and other modified BC reported. PEI-BC also showed good reusability in the adsorption of Cu(II) and Pb(II). This study demonstrates that polyethyleneimine modification makes BC a potential bioadsorbent for heavy metal ion removal in waste water.

In this study, an efficient, reusable, and environmental catalytic system consisting of sugarcane bagasse (an agricultural and sugar mill waste material, SCB) and KI was applied to the cycloaddition of carbon dioxide (CO2) to epoxides or aziridines under mild conditions for the first time. Their catalytic cycloaddition activities were found to be well correlated with the large quantities hydroxyl groups in SCB, which had a synergetic effect with the halide anion of KI. The as-prepared catalytic system also exhibited excellent cycloaddition activities for various epoxide or aziridine substrates as well. Moreover, the catalyst could be recovered and reused multiple times without obvious loss in activity. The present method represents an integrated and ideal green process for the utilization of biomass and “carbon neutral” resources, which has a high potential for large-scale fixation of CO2 into value-added chemicals.

The present study investigated the impact of regeneration process on the crystalline structure and enzymatic hydrolysis behaviors of microcrystalline cellulose (MCC) regenerated from ionic liquid 1-butyl-3-methylimidazolium chloride. The crystalline structures of these regenerated samples were analyzed by X-ray diffraction. Results suggested that almost amorphous cellulose was obtained by regenerating MCC in acetone (DRC-a), while partial cellulose II structure could be found in these regenerated samples from water and ethanol. Additionally, the enzymatic hydrolysis behaviors of MCC and its regenerated samples were comparatively studied. Results showed that above 90% of cellulose could be converted into glucose within 4 h for DRC-a and regenerated cellulose without drying (WRC-w) as compared to that of MCC (9.7%). Therefore, the regeneration process could significantly influence the crystallinity and digestibility of cellulose.

Lignocellulosic biomass in the form of plant materials offers the most abundant renewable resource in replacing traditional fossil resources. In the present study, lignocellulosic biomass films were prepared directly from bagasse solutions in DMSO/LiCl without additional film-forming additives by coagulation either in an acetone/water (9/1, v/v) mixture or in water following a freezing treatment. The physicochemical properties of bagasse films were studied by FT-IR, UV/vis, SEM, XRD, and tensile testing. The films were semi-transparent with a yellow color and showed strong abilities in UV light blocking due to the presence of lignin. Relatively high mechanical strength comparable to cellulose–starch–lignin composite films was observed. The physical cross-link and the stress buffer of micropores formed in the freezing treatment resulted in enhanced mechanical strength of the film. The facile and environmentally friendly process creates a new strategy for converting abundant lignocellulosic materials to nov...

Background: A bacterial β-etherase pathway uses glutathione-dependent enzymes for catabolism of β-ether-linked substructures found in lignin. Results: Racemic β-ether-linked substructures are stereoselectively converted to thioether-linked monoaromatic compounds by β-etherase pathway enzymes. Conclusion: Multiple enzymes with complementary stereochemical features are needed to catabolize racemic lignin substructures. Significance: Understanding the β-etherase pathway metabolism of lignin enhances our potential to use lignin for agricultural, industrial, and biotechnological purposes., Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.

Plant metabolomics is increasingly used for pathway discovery and to elucidate gene function. However, the main bottleneck is the identification of the detected compounds. This is more pronounced for secondary metabolites as many of their pathways are still underexplored. Here, an algorithm is presented in which liquid chromatography–mass spectrometry profiles are searched for pairs of peaks that have mass and retention time differences corresponding with those of substrates and products from well-known enzymatic reactions. Concatenating the latter peak pairs, called candidate substrate-product pairs (CSPP), into a network displays tentative (bio)synthetic routes. Starting from known peaks, propagating the network along these routes allows the characterization of adjacent peaks leading to their structure prediction. As a proof-of-principle, this high-throughput cheminformatics procedure was applied to the Arabidopsis thaliana leaf metabolome where it allowed the characterization of the structures of 60% of the profiled compounds. Moreover, based on searches in the Chemical Abstract Service database, the algorithm led to the characterization of 61 compounds that had never been described in plants before. The CSPP-based annotation was confirmed by independent MSn experiments. In addition to being high throughput, this method allows the annotation of low-abundance compounds that are otherwise not amenable to isolation and purification. This method will greatly advance the value of metabolomics in systems biology.

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (

Bacterial cellulose (BC) can be used to improve the physical properties of paper. However, previous studies have showed that the effectiveness of this improvement is impaired by the agglomeration of the disintegrated BC fibers. Effective dispersion of BC fibers is important to their reinforcing effects to paper products, especially those made of recycled fibers. In this study, carboxymethyl cellulose, xylan, glucomannan, cationized starch, and polyethylene oxide were used to improve the dispersion of BC fibers. With dispersed BC fibers, the paper made of recycled fiber showed improved dry tensile strength. The best improvement in dry tensile index was 4.2 N&middot, m/g or 12.7% up, which was obtained by adding BC fibers dispersed with glucomannan. Glucomannan had the highest adsorption onto BC fibers, i.e., 750 mg/g at 1000 mg/L concentration, leading to the best colloidal stability of BC fiber suspension that had no aggregation in 50 min at 0.1 weight ratio of glucomannan to BC. TEMPO-mediated oxidation of BC was effective in improving its colloidal stability, but not effective in improving the ability of BC fiber to enhance paper dry tensile index while the wet tensile index was improved from 0.89 N&middot, m/g to 1.59 N&middot, m/g, i.e., ~80% improvement.

In the present work, a new expansion pretreatment was used to fractionate agricultural wastes (rice straw and rice husk) into lignin and carbohydrates. The expansion pretreatment takes the advantages of extrusion and milder steam explosion. The lignin fractions obtained from expanded rice straw and rice husk by mild alkaline extraction were characterized by gel permeation chromatography (GPC), sugar analysis, 2D NMR and derivatization followed by reductive cleavage (DFRC). Results showed that the expansion pretreatment resulted in higher extraction yields of lignin from pretreated rice straw than that from untreated control. The results from molecular weight measurement and DFRC analysis suggested that the condensation reaction could take place between lignin units during the expansion processing. 2D NMR spectra showed that guaiacyl (G) and syringyl (S) units were predominant in the lignin fractions, and a small amount of p-hydroxyphenyl (H) unit was also detected. It was also found that the flavones-type structure tricin was apparently present in lignin fractions from rice straw but not in rice husk. Considering the high extraction yields and purity of lignins from pretreated biomass, the expansion pretreatment could be an attractive method for fractionation of agricultural wastes.

Catalytic oxidation via the application of molecular oxygen and copper complexes is a useful pathway toward valuable low molecular mass compounds from in situ or waste stream lignins. In this study, two dimeric β-ether model compounds, one β-ether oligomer, and a milled wood lignin sample from Loblolly pine were catalytically oxidized. Yields and stability of the aromatic aldehyde and acid products were measured. Nuclear magnetic resonance spectroscopy and gel permeation chromatography were used to monitor structure/composition and molecular mass changes of the lignin before and after catalytic oxidation to study the degree of depolymerization and structure of the residual lignin. Oxidized units appear to be derived from β-aryl ether, phenylcoumaran, and biphenyl ether components. To date, this method breaks down the lignin polymeric structure reasonably effectively, producing low molecular mass products; this work also highlights some of the issues that need to be overcome to optimize this approach.

Verticillium dahliae is an economically relevant soilborne pathogen that causes vascular wilt in several crops, including pepper (Capsicum annuum). Fungal infection is usually visualized as a vascular browning, likely due to the onset of phenylpropanoid metabolism, which also seems to play a crucial role in the tolerance of some pepper varieties. In the current work, the potential function of distinct phenylpropanoid derivatives (suberin, lignin and phenolic compounds) in the pepper tolerance response against V. dahliae, was investigated. Histochemical and biochemical analyses ruled out suberin as a key player in the pepper-fungus interaction. However, changes observed in lignin composition and higher deposition of bound phenolics in infected stems seemed to contribute to the reinforcement of cell walls and the impairment of V. dahliae colonization. Most importantly, this is the first time that the accumulation of the hydroxycinnamic acid amide N-feruloyltyramine was reported in pepper stems in response to a vascular fungus. Fungitoxic activity for that hydroxycinnamate-tyramine conjugate was demonstrated as well.

In the present study, we describe a novel one-step method to prepare water-soluble cellulose acetate (WSCA) with higher degree of polymerization values (DP = 650–680) by in situ activation of carboxyl group in ionic liquid. First of all, cellulose was dissolved in 1-ethyl-3-methylimidazolium acetate (EmimAc) and reacted with dichloroacetyl chloride (Cl2AcCl) in order to make cellulose dichloroacetate. Under various conditions, a series of water soluble products were produced. Elemental analysis and NMR results confirmed that they were cellulose acetate with DS (degree of substitution) values in the range from 0.30 to 0.63. NMR studies demonstrated that Cl2AcCl reacted with acetate anion of EmimAc producing a mixed anhydride that acetylated cellulose. Other acylating reagents such as benzoyl chloride, chloroacetyl chloride can also work similarly. 2D NMR characterization suggested that 6-mono-O-acetyl moiety, 3,6-di-O-acetylcellulose and 2,6-di-O-acetyl cellulose were all synthesized and the reactivity of hydroxyl groups in anhydro-glucose units was in the order C-6>C-3>C-2. This work provides an alternative way to make WSCA, meanwhile, also services as a reminder that the activity of EmimAc toward carbohydrate as acylating reagents could be a problem, because the expected acylated products may not be resulted and recycling of this ionic liquid could also be difficult.

12 páginas.-- 4 figuras.-- 3 tablas.-- 59 referencias.-- Additional Supporting Information may be found in the online version of this article. http://dx.doi.org/10.1111/tpj.13315, Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as an authentic monomer in grass lignification that likely functions as a nucleation site. It is linked onto lignin as an aryl alkyl ether by radical coupling with monolignols or their acylated analogs. However, the level of tricin that incorporates into lignin remains unclear. Herein, three lignin characterization methods: acidolysis; thioacidolysis; and derivatization followed by reductive cleavage; were applied to quantitatively assess the amount of lignin-integrated tricin. Their efficiencies at cleaving the tricin-(4′–O–β)-ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexadeuterated tricin analog was synthesized as an internal standard for accurate quantitation purposes. Thioacidolysis proved to be the most efficient method, liberating more than 91% of the tricin with little degradation. A survey of different seed-plant species for the occurrence and content of tricin showed that it is widely distributed in the lignin from species in the family Poaceae (order Poales). Tricin occurs at low levels in some commelinid monocotyledon families outside the Poaceae, such as the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the non-commelinid monocotyledon family Orchidaceae (Orchidales). One eudicotyledon was found to have tricin (Medicago sativa, Fabaceae). The content of lignin-integrated tricin is much higher than the extractable tricin level in all cases. Lignins, including waste lignin streams from biomass processing, could therefore provide a large and alternative source of this valuable flavone, reducing the costs, and encouraging studies into its application beyond its current roles., The authors thank the China Scholarship Council, State Education Department, for supporting living expenses for Wu Lan’s PhD Program in the Department of Biological System Engineering, University of Wisconsin, Madison, USA. WL, FL, SK and JRa were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). JR e was funded by the Spanish Project CTQ2014-60764-JIN (co-financed by FEDER funds), BGS and PJH by the University of Auckland, and JRa, BGS and PJH in part by US Department of Energy, Energy Biosciences Program, Grant #DE-AI02-06ER64299 (2006).

11 páginas.-- 5 figuras.-- 1 tabla.-- 47 referencias.-- The following supplemental materials are available in the https://doi.org/10.1104/pp.16.02012, Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed ‘candidate substrate product pair’ algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots. Lignin is one of the major components in plant cell walls and is deposited predominantly in the walls of secondarily thickened cells. It is a complex phenylpropanoid polymer composed primarily of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units derived from the monolignols p-coumaryl 2h, coniferyl 2g, and sinapyl 2s alcohols, respectively (Fig. 1; Freudenberg and Neish, 1968). These monolignols are biosynthesized in the cytoplasm and translocated to the cell wall, where they are oxidized by laccases and peroxidases to monolignol radicals (Boerjan et al., 2003; Dixon and Reddy, 2003; Ralph et al., 2004b; Vanholme et al., 2008, 2010; Bonawitz and Chapple, 2010; Mottiar et al., 2016). The polymer can be started by radical coupling between two monolignol radicals to form a dehydrodimer from which the chain extends by endwise polymerization with additional monolignols, producing β-O-4-, β-5-, β-1-, and β-β-linked units in the lignin. Two growing oligomers also may radically couple to increase the polymer size, producing 4-O-5- and 5-5-linked units. During such radical coupling reactions, therefore, the monomer-derived units are linked together via various C-C and C-O bonds with different frequencies depending primarily on the monomer distribution and supply (Ralph et al., 2004b)., This work was supported by the Department of Energy Great Lakes Bioenergy Research Center (grant no. DE–FC02–07ER64494 to W.L., F.L., and J.R.), by Stanford University’s Global Climate and Energy Program (to J.R., K.M., and W.B.), by the Institute for the Promotion of Innovation through Science and Technology in Flanders via the SBO project Bioleum (to W.B. and K.M.), by the China Scholarship Council's State Education Department for his Ph.D. work in the Department of Biological System Engineering, University of Wisconsin, Madison (to W.L.).

Plants have convergently evolved to use monolignol ferulate conjugates to produce lignins containing chemically labile backbone esters., Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl–coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.

Wood shortages, environmental pollution and high energy consumption remain major obstacles hindering the development of today’s pulp and paper industry. Energy-saving and environmental friendly pulping processes are still needed, especially for non-woody materials. In this study, soda-oxygen pulping of bagasse was investigated and a successful soda-oxygen pulping process for bagasse at 100 °C was established. The pulping parameters of choice were under active alkali charge of 23%, maximum cooking temperature 100 °C, time hold at maximum temperature 180 min, initial pressure of oxygen 0.6 MPa, MgSO4 charge 0.5%, and de-pithed bagasse consistency 12%. Properties of the resultant pulp were screened yield 60.9%, Kappa number 14, viscosity 766 dm3/kg, and brightness 63.7% ISO. Similar pulps were also obtained at 110 °C or 105 °C with a cooking time of 90 min. Compared with pulps obtained at higher temperatures (115–125 °C), this pulp had higher screened yield, brightness, and acceptable viscosity, while the delignification degree was moderate. These results indicated that soda-oxygen pulping at 100 °C, the lowest cooking temperature reported so far for soda-oxygen pulping, is a suitable process for making chemical pulp from bagasse. Pulping at lower temperature and using oxygen make it an environmental friendly and energy-saving pulping process.

Recent advances in nuclear magnetic resonance (NMR) technology have made it possible to rapidly screen plant material and discern whole cell wall information without the need to deconstruct and fractionate the plant cell wall. This approach can be used to improve our understanding of the biology of cell wall structure and biosynthesis, and as a tool to select plant material for the most appropriate industrial applications. This is particularly true in an era when renewable materials are vital to the emerging bio-based economies. This protocol describes procedures for (i) the preparation and extraction of a biological plant tissue, (ii) solubilization strategies for plant material of varying composition and (iii) 2D NMR acquisition (for typically 15 min-5 h) and integration methods used to elucidate lignin subunit composition and lignin interunit linkage distribution, as well as cell wall polysaccharide profiling. Furthermore, we present data that demonstrate the utility of this new NMR whole cell wall characterization procedure with a variety of degradative methods traditionally used for cell wall compositional analysis.

Analytical thioacidolysis releases diagnostic monomers from lignins by selectively cleaving alkyl aryl ether bonds. High yielding syntheses of the three thioacidolysis monomers were developed, and the compounds were used as standards for gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID) quantitation of monomers released from lignocellulosics. First, syringyl, guaiacyl, and p-hydroxyphenyl glycerols were synthesized from the corresponding ethyl cinnamates and used as thioacidolysis substrates to prepare the monomers in high yields. These monomers were then used as standard compounds to measure their GC-MS and GC-FID response factors against two internal standards, 4,4'-ethylenebisphenol and tetracosane. For quantitation, it was shown that 4,4'-ethylenebisphenol is the better internal standard for GC-MS, whereas tetracosane remains the choice for GC-FID. When the obtained response factors were applied, the thioacidolysis monomer yields from white spruce, loblolly pine, poplar, bamboo, and sugar cane bagasse were determined by GC-MS. The obtained results were consistent with those reported in the literature.

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies.

Underwater vibration detection is of great importance in personal safety, environmental protection, and military defense. Sealing layers are required in many underwater sensor architectures, leading to limited working-life and reduced sensitivity. Here, a flexible, superhydrophobic, and conductive tungsten disulfide (WS2) nanosheets-wrapped sponge (SCWS) is reported for the high-sensitivity detection of tiny vibration from the water surfaces and from the grounds. When the SCWS is immersed in water, a continuous layer of bubbles forms on its surfaces, providing the sensor with two special abilities. One is sealing-free feature due to the intrinsic water-repellent property of SCWS. The other is functioning as a vibration-sensitive medium to convert mechanical energy into electric signals through susceptible physical deformation of bubbles. Therefore, the SCWS can be used to precisely detect tiny vibration of water waves, and even sense those caused by human footsteps, demonstrating wide applications of this amphibious (water/ground) vibration sensor. Results of this study can initiate the exploration of superhydrophobic materials with elastic and conductive properties for underwater flexible electronic applications.

Although the primary structure of proteins, nucleic acids, and carbohydrates can be readily determined, no sequencing method has been described yet for the second most abundant biopolymer on earth (i.e. lignin). Within secondary-thickened plant cell walls, lignin forms an aromatic mesh arising from the combinatorial radical-radical coupling of monolignols and many other less abundant monomers. This polymerization process leads to a plethora of units and linkage types that affect the physicochemical characteristics of the cell wall. Current methods to analyze the lignin structure focus only on the frequency of the major monomeric units and interunit linkage types but do not provide information on the presence of less abundant unknown units and linkage types, nor on how linkages affect the formation of neighboring linkages. Such information can only be obtained using a sequencing approach. Here, we describe, to our knowledge for the first time, a sequencing strategy for lignin oligomers using mass spectrometry. This strategy was then evaluated on the oligomers extracted from wild-type poplar (Populus tremula × Populus tremuloides) xylem. In total, 134 lignin trimers to hexamers were observed, of which 36 could be completely sequenced. Interestingly, based on molecular mass data of the unknown monomeric and dimeric substructures, at least 10 unknown monomeric units or interunit linkage types were observed, one of which was identified as an arylglycerol end unit.

Grasses are a predominant source of nutritional energy for livestock systems around the world. Grasses with high lignin content have lower energy conversion efficiencies for production of bioenergy either in the form of ethanol or to milk and meat through ruminants. Grass lignins are uniquely acylated with p-coumarates (pCA), resulting from the incorporation of monolignol p-coumarate conjugates into the growing lignin polymer within the cell wall matrix. The required acyl-transferase is a soluble enzyme (p-coumaroyl transferase, pCAT) that utilizes p-coumaroyl-CoenzymeA (pCA-CoA) as the activated donor molecule and sinapyl alcohol as the preferred acceptor molecule. Grasses (C3and C4) were evaluated for cell wall characteristics; pCA, lignin, pCAT activity, and neutral sugar composition. All C3 and C4 grasses had measurable pCAT activity, however the pCAT activities did not follow the same pattern as the pCA incorporation into lignin as expected.

BACKGROUND: We used a biomimetic model system to ascertain how reductions in ferulate – lignin cross-linking and shifts in lignin composition influence ruminal cell wall fermentation. Primary walls from maize cell suspensions with normal or reduced feruloylation were artificially lignified with various monolignols previously identified in normal, mutant, and transgenic plants. Cell wall fermentability was determined from gas production during invitro incubation with rumen microflora and by analysis of non-fermented polysaccharides. RESULTS: Hemicellulose fermentation lag time increased by 37%, rate decreased by 37%, and the extent declined by 18% as cell wall lignin content increased from 0.5 to 124 mg g −1 . Lignification increased lag time for cellulose fermentation by 12-fold. Ferulate – lignin cross-linking accounted for at least one-half of the inhibitory effect of lignin on cell wall fermentation. Incorporating sinapyl p-coumarate, a precursor of p-coumaroylated grass lignin, increased the extent of hemicellulose fermentation by 5%. Polymerizing varying ratios of coniferyl and sinapyl alcohols or incorporating 5-hydroxyconiferyl alcohol, coniferaldehyde, sinapyl acetate, or dihydroconiferyl alcohol into lignin did not alter the kinetics of cell wall fermentation. CONCLUSION: The results indicate that selection or engineering of plants for reduced lignification or ferulate – lignin cross-linking will improve fiber fermentability more than current approaches for shifting lignin composition. c � 2008 Society of Chemical Industry

Biodegradation by brown-rot fungi is quantitatively one of the most important fates of lignocellulose in nature. It has long been thought that these basidiomycetes do not degrade lignin significantly, and that their activities on this abundant aromatic biopolymer are limited to minor oxidative modifications. Here we have applied a new technique for the complete solubilization of lignocellulose to show, by one-bond (1)H-(13)C correlation nuclear magnetic resonance spectroscopy, that brown rot of spruce wood by Gloeophyllum trabeum resulted in a marked, non-selective depletion of all intermonomer side-chain linkages in the lignin. The resulting polymer retained most of its original aromatic residues and was probably interconnected by new linkages that lack hydrogens and are consequently invisible in one-bond (1)H-(13)C correlation spectra. Additional work is needed to characterize these linkages, but it is already clear that the aromatic polymer remaining after extensive brown rot is no longer recognizable as lignin.

*Summary A molecular marker compound, derived from lignin by the thioacidolysis degradative method, for structures produced when ferulic acid is incorporated into lignin in angiosperms (poplar, Arabidopsis, tobacco), has been structurally identified as 1,2,2-trithioethyl ethylguaiacol [1-(4-hydroxy-3-methoxyphenyl)-1,2,2-tris(ethylthio)ethane]. Its truncated side chain and distinctive oxidation state suggest that it derives from ferulic acid that has undergone bis-8-O-4 (cross) coupling during lignification, as validated by model studies. A diagnostic contour for such structures is found in two-dimensional 13 C‐ 1 H correlated (HSQC) NMR spectra of lignins isolated from cinnamoyl CoA reductase (CCR)-deficient poplar. As low levels of the marker are also released from normal (i.e. non-transgenic) plants in which ferulic acid may be present during lignification, notably in grasses, the marker is only an indicator for CCR deficiency in general, but is a reliable marker in woody angiosperms such as poplar. Its derivation, together with evidence for 4-O-etherified ferulic acid, strongly implies that ferulic acid is incorporated into angiosperm lignins. Its endwise radical coupling reactions suggest that ferulic acid should be considered an authentic lignin precursor. Moreover, ferulic acid incorporation provides a new mechanism for producing branch points in the polymer. The findings sharply contradict those reported in a recent study on CCR-deficient Arabidopsis.

Grass cell walls are atypical because their xylans are acylated with ferulate and lignins are acylated with p-coumarate. To probe the role and interactions of these p-hydroxycinnamates during lignification, feruloylated primary cell walls isolated from maize cell suspensions were lignified with coniferyl and sinapyl alcohols and with varying levels of p-coumarate esters. Ferulate xylan esters enhanced the formation of wall-bound syringyl lignin more than methyl p-coumarate, however, maximal concentrations of syringyl lignin were only one-third that of guaiacyl lignin. Including sinapyl p-coumarate, the presumed precursor of p-coumaroylated lignins, with monolignols unexpectedly accelerated peroxidase inactivation, interfered with ferulate copolymerization into lignin, and had minimal or adverse effects on cell wall lignification. Free phenolic groups of p-coumarate esters in isolated maize lignin and pith cell walls did not undergo oxidative coupling with each other or with added monolignols. Thus, the extensive formation of syringyl-rich lignins and the functional role of extensive lignin acylation by p-coumarate in grasses remains a mystery.

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Two major residues are produced by the sugarcane industry, the fibrous fraction following juice extraction (bagasse), and the harvest residue (straw). The structures of the lignins from these residues were studied by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). Whereas the lignin from bagasse has a syringyl-rich p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) molar composition of 2:38:60, the lignin from straw is guaiacyl-rich (H:G:S of 4:68:28). The compositional differences were also reflected in the relative abundances of the different interunit linkages. Bagasse lignin was primarily β–O–4′ alkyl-aryl ether units (representing 83% of NMR-measurable units), followed by minor amounts of β–5′ (phenylcoumarans, 6%) and other condensed units. The lignin from straw has lower levels of β-ethers (75%) but higher relative levels of phenylcoumarans (β–5′, 15%) and dibenzodioxocins (5–5/4–O–β, 3%), consistent with a lignin enriched in G-units. Both lignins are extensively acylated at the γ-hydroxyl of the lignin side-chain (42% and 36% acylation in bagasse and straw), predominantly with p-coumarates (preferentially on S-units) but also with acetates (preferentially on G-units) to a minor extent. Tetrahydrofuran structures diagnostically arising from β–β-coupling (dehydrodimerization) of sinapyl p-coumarate or its cross-coupling with sinapyl alcohol were found in both lignins, indicating that sinapyl p-coumarate acts as a monomer participating in lignification. The flavone tricin was also found in the lignins from sugarcane, as also occurs in other grasses., This study has been funded by the Spanish projects AGL2011-25379, AGL2014-53730-R and CTQ2014-60764-JIN (co-financed by FEDER funds), the CSIC project 2014-40E-097 and the EU-projects LIGNODECO (KBBE-2009-3-244362) and INDOX (KBBE-2013-7-613549). John Ralph was funded through the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). Jorge Rencoret thanks the CSIC for a JAE-DOC contract of the program “Junta para la Ampliación de Estudios” co-financed by Fondo Social Europeo (FSE). A.G. Lino thanks CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for financial support. C.F. Lima and J.L. Colodette are grateful to National Council for Scientific and Technological Development (CNPq) for research fellowships. We also thank Dr. Manuel Angulo for performing the NMR analyses that were acquired on a Bruker Avance III 500 MHz instrument from the NMR facilities of the General Research Services of the University of Seville (SGI-CITIUS).

Spirodienone structures have been detected in spruce, birch, and kenaf lignin isolates. NMR signals corresponding to guaiacyl and syringyl spirodienones were fully identified and assigned based on 13C, QUAT, HSQC, HSQC‐TOCSY, and HMBC NMR data. Spruce lignin contains spirodienone structures of the guaiacyl type. Syringyl spirodienones dominate in kenaf and birch lignins. Each type of spirodienone was found to be present in two different stereoisomeric forms, with one of the isomers being more prevalent. Signal integrations indicate that about three spirodienones per 100 phenylpropanoid units are present in the spruce and birch lignins and about four in the kenaf lignin.

A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically derives from 8-8-coupling of ferulate followed by water addition to one of the incipient quinone methide moieties and internal trapping of the other. The finding adds complexity to the analysis of plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways occurring in planta.