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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. [ABSTRACT FROM AUTHOR]

Pyrolytic lignin and hydrogenated pyrolytic lignin were characterized by 2D 1H–13C HSQC and quantitative 13C NMR techniques. The pyrolytic lignin was produced from a mixed maple wood feedstock and separated from the bio-oil by water extraction. p-Hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatics were the basic units of pyrolytic lignin. The native lignin β-aryl ether, phenylcoumaran and resinol structures were not present in the pyrolytic lignin. The hydrogenation was conducted with a Ru/TiO2 catalyst at temperatures ranging from 25–150 °C with higher temperatures exhibiting higher levels of hydrogenation. Solid coke formed on the catalyst surface (1% coke yield) even for hydrogenation at 25 °C. The carbon yield of pyrolytic lignin to coke increased from 1% to 5% as the hydrogenation temperature increased from 25 to 150 °C. A single-step hydrogenation at 150 °C resulted in a reduction from 65% to 39% aromatic carbons. A three-step hydrogenation scheme at this same temperature resulted in a reduction of aromatic carbons from 65% to 17%. The decrease in the aromatic carbon corresponded with an increase in the aliphatic carbon. Coke formation reduced from a 5% carbon yield of pyrolytic lignin in the first hydrogenation step to a 1% carbon yield in each of the second and third hydrogenation steps. The pyrolytic lignin could be separated into a high and low molecular weight fraction. The coke yield from the high molecular weight fraction was twice as much as that from the low molecular weight fraction. [ABSTRACT FROM AUTHOR]

Background: Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has previously been determined to substantially improve the enzymatic hydrolysis of woody plants including hybrid poplar as a consequence of moderate delignification. In the present work, cell wall morphological and lignin structural changes were characterized for this pretreatment approach to gain insights into pretreatment outcomes and, specifically, to identify the extent and nature of lignin modification. Results: Through TEM imaging, this catalytic oxidation process was shown to disrupt cell wall layers in hybrid poplar. Cu-containing nanoparticles, primarily in the Cu(I) oxidation state, co-localized with the disrupted regions, providing indirect evidence of catalytic activity whereby soluble Cu(II) complexes are reduced and precipitated during pretreatment. The concentration of alkali-soluble polymeric and oligomeric lignin was substantially higher for the Cu-catalyzed oxidative pretreatment. This alkali-soluble lignin content increased with time during the catalytic oxidation process, although the molecular weight distributions were unaltered. Yields of aromatic monomers (including phenolic acids and aldehydes) were found to be less than 0.2 % (wt/wt) on lignin. Oxidation of the benzylic alcohol in the lignin side-chain was evident in NMR spectra of the solubilized lignin, whereas minimal changes were observed for the pretreatment-insoluble lignin. Conclusions: These results provide indirect evidence for catalytic activity within the cell wall. The low yields of ligninderived aromatic monomers, together with the detailed characterization of the pretreatment-soluble and pretreatment- insoluble lignins, indicate that the majority of both lignin pools remained relatively unmodified. As such, the lignins resulting from this process retain features closely resembling native lignins and may, therefore, be amenable to subsequent valorization. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate in vitro antioxidant, NO scavenging, and antibacterial activities as well as total phenolic contents of different extracts of flowers of Jasminum humile and roots of Dorema aucheri. The plant extracts showed significant antioxidant activity, having IC50 values comparable to those of references used in each assay and also inhibited accumulation of nitrite in vitro. The plant extracts yielded phenolic contents and showed significant antibacterial activity. The observed antioxidant potential and phenolic contents of the extracts showed that flowers of J. humile and roots of D. aucheri are potential source of natural antioxidants that may help to retard oxidative degradation and microbial growth in food industry. [ABSTRACT FROM AUTHOR]

Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of [M + H]+ ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX™)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals. [ABSTRACT FROM AUTHOR]

An efficient organocatalytic method for chemoselective aerobic oxidation of secondary benzylic alcohols within lignin model compounds has been identified. Extension to selective oxidation in natural lignins has also been demonstrated. The optimal catalyst system consists of 4-acetamido-TEMPO (5 mol %; TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl) in combination with HNO3 and HCl (10 mol % each). Preliminary studies highlight the prospect of combining this method with a subsequent oxidation step to achieve C–C bond cleavage. [ABSTRACT FROM AUTHOR]

We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chrysosporium gene expression patterns are substantially influenced by lignin composition. [ABSTRACT FROM AUTHOR]

Background: Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has previously been determined to substantially improve the enzymatic hydrolysis of woody plants including hybrid poplar as a consequence of moderate delignification. In the present work, cell wall morphological and lignin structural changes were characterized for this pretreatment approach to gain insights into pretreatment outcomes and, specifically, to identify the extent and nature of lignin modification., Results: Through TEM imaging, this catalytic oxidation process was shown to disrupt cell wall layers in hybrid poplar. Cu-containing nanoparticles, primarily in the Cu(I) oxidation state, co-localized with the disrupted regions, providing indirect evidence of catalytic activity whereby soluble Cu(II) complexes are reduced and precipitated during pretreatment. The concentration of alkali-soluble polymeric and oligomeric lignin was substantially higher for the Cu-catalyzed oxidative pretreatment. This alkali-soluble lignin content increased with time during the catalytic oxidation process, although the molecular weight distributions were unaltered. Yields of aromatic monomers (including phenolic acids and aldehydes) were found to be less than 0.2 % (wt/wt) on lignin. Oxidation of the benzylic alcohol in the lignin side-chain was evident in NMR spectra of the solubilized lignin, whereas minimal changes were observed for the pretreatment-insoluble lignin., Conclusions: These results provide indirect evidence for catalytic activity within the cell wall. The low yields of lignin-derived aromatic monomers, together with the detailed characterization of the pretreatment-soluble and pretreatment-insoluble lignins, indicate that the majority of both lignin pools remained relatively unmodified. As such, the lignins resulting from this process retain features closely resembling native lignins and may, therefore, be amenable to subsequent valorization.

Dehydrodimerization of ferulates in grass cell walls provides a pathway toward cross-linking polysaccharide chains limiting the digestibility of carbohydrates by ruminant bacteria and in general affecting the utilization of grass as a renewable bioresource. Analysis of dehydrodiferulates (henceforth termed diferulates) in plant cell walls is useful in the evaluation of the quality of dairy forages as animal feeds. Therefore, there has been considerable demand for quantities of diferulates as standards for such analyses. Described here are syntheses of diferulates from ethyl ferulate via biomimetic radical coupling reactions using the copper(II)-tetramethylethylenediamine [CuCl(OH)-TMEDA] complex as oxidant or catalyst. Although CuCl(OH)-TMEDA oxidation of ethyl ferulate in acetonitrile produced mixtures composed of 8-O-4-, 8-5-, 8-8- (cyclic and noncyclic), and 5-5-coupled diferulates, a catalyzed oxidation using CuCl(OH)-TMEDA as catalyst and oxygen as an oxidant resulted in better overall yields of such diferulates. Flash chromatographic fractionation allowed isolation of 8-8- and 5-5-coupled diferulates. 8-5-Diferulate coeluted with 8-O-4-diferulate but was separated from it via crystallization; the 8-O-4 diferulate left in the mother solution was isolated by rechromatography following a simple tetrabutylammonium fluoride treatment that converted 8-5-diferulate to another useful diferulate, 8-5-(noncyclic) diferulate. Therefore, six of the nine (5-5, 8-O-4, 8-5-c, 8-5-nc, 8-5-dc, 8-8-c, 8-8-nc, 8-8-THF, 4-O-5) diferulic acids that have to date been found in the alkaline hydrolysates of plant cell walls can be readily synthesized by the CuCl(OH)-TMEDA catalyzed aerobic oxidative coupling reaction and subsequent saponification described here.

Lignocellulosic materials derived from forages and agricultural residues are potential sustainable resources for production of bioethanol or other liquid biofuels. However, the natural recalcitrance of such materials to enzymatic hydrolysis is a major obstacle in their efficient utilization. In grasses, much of the recalcitrance is associated with ferulate cross-linking in the cell wall, i.e., with polysaccharide-polysaccharide cross-linking that results from ferulate dehydrodimerization or with lignin-polysaccharide cross-linking that results from the incorporation of (polysaccharide-bound) ferulates or diferulates into lignin, mainly via free-radical coupling reactions. Many pretreatment methods have been developed to address recalcitrance, with ammonia pretreatments in general, and the AFEX (Ammonia Fiber Expansion) process in particular, among the more promising methods. In order to understand the polysaccharide liberating reactions involved in the cleavage of diferulate cell wall cross-links during AFEX pretreatment, reaction products from five esters modeling the major diferulates in grass cell walls treated under AFEX-like conditions were separated and characterized by NMR and HR-MS. Results from this study indicate that, beyond the anticipated amide products, a range of degradation products derive from an array of cleavage and substitution reactions, and reveal various pathways for incorporating ammonia-based nitrogen into biomass.