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Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor , alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS., Competing Interests: The authors declare no competing financial interest.

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 13 C 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, 12 C and 13 C lignin were isolated from nonlabeled and uniformly 13 C 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, 13 C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12 C lignin analogue and was shown to be extremely accurate (>99.9%, R 2 > 0.999) and precise (RSD < 1.5%). Third, 13 C-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.

Eucalyptus globulus wood samples were reacted in aqueous media (hydrothermal treatments) at 160 degrees C for 30-66 min. Liquors from the several experiments were analyzed by spectrophotometry, high-performance liquid chromatography, or gas chromatography-mass spectrometry for monosaccharides, oligosaccharides, oligosaccharide substituents (arabinose moieties, uronic acids, and acetyl groups), acetic acid, furfural, hydroxymethylfurfural, and dichloromethane-soluble compounds. Individual components of this latter fraction were identified and quantified. The molecular weight distribution of oligosaccharides was studied by high-performance size exclusion chromatography. The kinetics of xylan conversion into high-, medium-, and low-molecular-weight products was assessed in terms of the severity factor and by pseudohomogeneous kinetic models.

Xylo-oligosaccharides (XOS) with various substituents were fermented in vitro by fecal inocula (FI) from four human volunteers to study the influence of substitution on the ability and rate of fermentation and on the production of short-chain fatty acids (SCFA) and lactate. By all FI used nonsubstituted XOS (nXOS) and arabino-XOS (AXOS) were fermented more quickly than the more complex structures of acetylated XOS (AcXOS) and XOS containing a 4-O-methylglucuronic acid group (GlcA(me)XOS). In the first stage (0-40 h) of the fermentations of nXOS and AXOS mainly acetate and lactate were formed. The fermentations of AcXOS and GlcA(me)XOS resulted in a lower lactate production, whereas the concentration of propionate and butyrate increased. These results put emphasis on the detailed elucidation of the structural features of nondigestible oligosaccharides in general to understand their fermentation mechanisms more precisely.