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Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2> 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans.

Lignin is a highly abundant aromatic polymer in nature, but its controlled cleavage or cross-linking is a major challenge and currently hindering industrial applicability. Laccase (L) and laccase/mediator systems (LMS) are promising tools for enzymatic lignin modification, but to date, their overall reaction outcome is hard to predict and control. This research aimed to understand the reactivity of native and sulfonated β-O-4 linked lignin structures in L and LMS treatments. Trametes versicolorlaccase, and the mediator hydroxybenzotriazole (HBT) were used, and reaction products were analyzed using UHPLC-MSnand MALDI-TOF-MS. Polymerization was observed for both the native and sulfonated phenolic compounds, suggesting that sulfonation does not affect radical coupling of the phenolic lignin subunits. In contrast, sulfonation of the non-phenolic lignin structure prevented Cαoxidation and cleavage by L/HBT, which was explained by an increased Cα–H bond dissociation energy of ∼10 kcal mol−1upon sulfonation. Overall, our results indicate that lignin sulfonation drives the overall outcome of LMS incubations towards polymerization.