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Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive.

Miscanthus is a perennial grass suitable for the production of lignocellulosic biomass on marginal lands. The effects of salt stress on Miscanthus cell wall composition and its consequences on biomass quality have nonetheless received relatively little attention. In this study, we investigated how exposure to moderate (100 mM NaCl) or severe (200 mM NaCl) saline growing conditions altered the composition of both primary and secondary cell wall components in the stems of 15 Miscanthus sinensis genotypes. The exposure to stress drastically impacted biomass yield and cell wall composition in terms of content and structural features. In general, the observed compositional changes were more pronounced under severe stress conditions and were more apparent in genotypes with a higher sensitivity towards stress. Besides a severely reduced cellulose content, salt stress led to increased pectin content, presumably in the form of highly branched rhamnogalacturonan type I. Although salt stress had a limited effect on the total lignin content, the acid-soluble lignin content was strongly increased in the most sensitive genotypes. This effect was also reflected in substantially altered lignin structures and led to a markedly reduced incorporation of syringyl subunits and p-coumaric acid moieties. Interestingly, plants that were allowed a recovery period after stress ultimately had a reduced lignin content compared to those continuously grown under control conditions. In addition, the salt stress-induced cell wall alterations contributed to an improved enzymatic saccharification efficiency.

Understanding the structure of hardwoods can permit better valorization of lignin by enabling the optimization of green, high-yield extraction protocols that preserve the structure of wood biopolymers. To that end, a mild protocol was applied for the extraction of lignin from ball-milled birch. This made it possible to understand the differences in the extractability of lignin in each extraction step. The fractions were extensively characterized using 1D and 2D nuclear magnetic resonance spectroscopy, size exclusion chromatography, and pyrolysis-gas chromatography-mass spectrometry. This comprehensive characterization highlighted that lignin populations extracted by warm water, alkali, and ionic liquid/ethanol diverged in structural features including subunit composition, interunit linkage content, and the abundance of oxidized moieties. Moreover, ether- and ester-type lignin-carbohydrate complexes were identified in the different extracts. Irrespective of whether natively present in the wood or artificially formed during extraction, these complexes play an important role in the extractability of lignin from ball-milled hardwood. Our results contribute to the further improvement of lignin extraction strategies, for both understanding lignin as present in the lignocellulosic matrix and for dedicated lignin valorization efforts., QC 20231130

Fungal pretreatment of plant biomass is often assessed by using single plant genotypes and single fungal strains, but can the process be improved by harmonizing both, thus selecting specific substrate-fungus combinations? To tackle this question, we treated four Miscanthus sinensis genotypes with four Ceriporiopsis subvermispora strains and thoroughly analyzed substrates and treated residues. The M. sinensis genotypes differed in cellulose, hemicellulose, and lignin contents and lignin-wise diverged in subunit and linkage composition and the incorporation of hydroxycinnamic acids and tricin. Independently of the M. sinensis genotype used, C. subvermispora strain MES13904 outperformed the other three strains in extent and selectivity of delignification and consistently generated the highest enzymatic residual carbohydrate conversion and structural changes in the residual lignin. The “best” substrate-fungus combination gave 63% w/w delignification and a total enzymatic glucose yield of 66% w/w, while the “worst” combination led to 3% w/w lignin removal only and negligible glucose yield improvement. Our study highlights that white-rot fungal treatment of plant biomass is driven by both compositional and structural features of the substrate as well as the genetic makeup of the fungal strain used. These insights contribute to expediting the biological valorization of lignocellulose and ultimately to enabling more controlled fungal pretreatments.

A first systematic approach on the base catalyzed depolymerization (BCD) of five technical lignins derived from various botanical origins (herbaceous, hardwood and softwood) and covering the main three industrial pulping methods (soda, kraft and organosolv) is reported. This study provides a first of its kind in-depth quantification and structural characterization of two main BCD fractions namely lignin oil and lignin residue, describing the influence of the BCD process conditions. Depolymerization is evaluated in terms of lignin conversion, lignin oil yield, phenolic monomer selectivity and the production of lignin residue and char. Lignin oils were extensively characterized by size exclusion chromatography (SEC), GC-MS, GC-FID, 13C-NMR, HSQC NMR and elemental analysis. GC × GC-FID was used to identify and quantify distinct groups of monomeric compounds (methoxy phenols, phenols, dihydroxy-benzenes) in the lignin oil. The lignin oil yields (w/w) ranged from 20-31% with total monomer contents ranging from 48 to 57% w/w. SEC analysis indicated the presence of dimers/oligomers in the lignin oil, which through HSQC NMR analysis were confirmed to contain new, non-native interunit linkages. 13C NMR analyses of the lignin oils suggest the presence of diaryl type linkages (i.e. aryl-aryl, aryl C-O) evidencing deconstruction and recombination of lignin fragments during BCD. Irrespective of the lignin source, a residue, often regarded as ‘unreacted’ residual lignin was the main product of BCD (43 to 70% w/w). Our study highlights that this residue has different structural properties and should not be considered as unreacted lignin, but rather as an alkali soluble condensed aromatic material. HSQC, DEPT-135, 13C, and 31P NMR and SEC analyses confirm that the BCD residues are indeed more condensed, with increased phenolic hydroxyl content and lower molecular weights compared to all feed lignins. Subsequent BCD of solid residual fractions produced only low oil yields (6-9% w/w)

Extraction of polyphenols from Centranthus longiflorus stems was conducted using ultrasound and infrared Ired-Irrad® techniques, and compared to the conventional water bath method. Response surface methodology was used to analyse the effect of time, temperature, and ethanol percentage, as well as to optimize the three extraction methods. The highest phenolic content (81 mg GAE/g DM) and antioxidant activity (76% DPPH inhibition) were recorded with the Ired-Irrad® extract obtained under the optimal conditions: 55 °C, 127 min, 48% (v/v) ethanol. Biological activities (antioxidant, antibacterial and antibiofilm) of the three extracts were assessed. All C. longiflorus stems extracts showed limited antibacterial effects regardless of the extraction method (MIC = 50 mg/mL), whereas Ired-Irrad® extract exhibited the highest biofilm eradication and prevention capacities (93% against Escherichia coli and 97% against Staphylococcus epidermidis, respectively). This bioactivity is likely related to abundant caffeoylquinic acid and quercetin rutinoside, as identified by RP-UHPLC-PDA-MS analysis. The results obtained further promote the effectiveness of Ired-Irrad® as a highly flexible and cost-efficient extraction technique.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello-oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H218O, it was confirmed that the C6 gem-diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem-diol, could not be excluded. These findings can be extended to how the reactive LPMO-cosubstrate complex is positioned towards the substrate.

Lignocellulosic crops are attractive bioresources for energy and chemicals production within a sustainable, carbon circular society. Miscanthus is one of the perennial grasses that exhibits great potential as a dedicated feedstock for conversion to biobased products in integrated biorefineries. The current biorefinery strategies are primarily focused on polysaccharide valorization and require severe pretreatments to overcome the lignin barrier. The need for such pretreatments represents an economic burden and impacts the overall sustainability of the biorefinery. Hence, increasing its efficiency has been a topic of great interest. Inversely, though pretreatment will remain an essential step, there is room to reduce its severity by optimizing the biomass composition rendering it more exploitable. Extensive studies have examined the miscanthus cell wall structures in great detail, and pinpointed those components that affect biomass digestibility under various pretreatments. Although lignin content has been identified as the most important factor limiting cell wall deconstruction, the effect of polysaccharides and interaction between the different constituents play an important role as well. The natural variation that is available within different miscanthus species and increased understanding of biosynthetic cell wall pathways have specified the potential to create novel accessions with improved digestibility through breeding or genetic modification. This review discusses the contribution of the main cell wall components on biomass degradation in relation to hydrothermal, dilute acid and alkaline pretreatments. Furthermore, traits worth advancing through breeding will be discussed in light of past, present and future breeding efforts.

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

Ammonia treatment of lignocellulose improves carbohydrate degradability, however, low ammonia dose treatment effects and mechanisms are hardly considered. This study describes low dose ammonia treatment of wheat straw in a statistical design of experiments (Taguchi design) to evaluate the effects of ammonia concentration, treatment time and the Solid:Liquid ratio on structure, composition and enzymatic degradability of the residual fractions. The results showed that low ammonia concentration (≤2 w/w % NH3) resulted in a high carbohydrate recovery (>80%) coupled enzymatic hydrolysis of 50% of xylan and 40% of glucan of the treated material using a (hemi-) cellulase enzyme cocktail. This effect coincidences with the relative decrease in ferulic acid by 10% and coumaric acid by more than 50% analysed via pyrolysis-GC–MS, measured as 4-vinyl-phenol and 4-vinyl-guaiacol, respectively. Our findings show that lowering ammonia concentration increased the effect of treatment time on the enzymatic degradability of the residual fraction.

BACKGROUND: White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal-treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py-GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS: Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of −0.84 versus −0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents – among others, due to interference of fungal biomass. CONCLUSION: Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes.

With the ever-advancing lignocellulose valorization strategies, lignin analyses need to advance as well. However, lignin quantification still heavily relies on unspecific, time- and sample-consuming gravimetric, and spectrophotometric analyses. Here, we demonstrate that lignin isolates from uniformly 13C-labeled wheat straw, willow, and douglas fir serve as "ideal" internal standards for pyrolysis gas chromatography high-resolution mass spectrometry (py-GC-HR-MS) analyses of plant biomass, allowing the accurate and precise quantification and structural characterization of lignin in grasses, hardwoods, and softwoods. The 13C lignin internal standards were comprehensively structurally characterized by HSQC NMR and py-GC-HR-MS analyses, and their application for lignin quantification was validated in biomass model systems and in actual plant biomass. For all botanical origins and species, the lignin content was determined within 5% relative deviation of the Klason benchmark. These results establish the capability of the developed analytical platform to selectively quantify and structurally characterize lignin simultaneously and demonstrate a valuable addition to the lignin analysis toolbox.