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Recent droughts have accentuated problems with attacks of the European spruce bark beetle (SBB) (Ips typographus) on Norway spruce (Picea abies), one of the most important tree species in European forestry. SBB attacks are typically accompanied by infestation with Ophiostomatoid fungi causing blue stain. The chemical composition of sapwood and bark from spruce trees infested by SBB, or by both SBB and blue-stain fungi (BSF), was investigated and compared to corresponding fractions from non-infested reference (REF) trees. While sapwood from infested trees showed higher carbohydrate:lignin (C:L) ratios (SBB, 2.21; BSF, 2.47) than sapwood from non-infested trees (REF, 2.17), BSF bark showed lower C:L ratio (0.81) than REF bark (1.23). For BSF sapwood and bark, and SBB bark, the fractions of extractives were half or less than half of that of the corresponding REF materials. Group analysis using GC-FID showed significantly (p ≤ 0.01) lower levels of resin acids, fatty acids, steryl esters, and triglycerides in BSF materials than in REF materials, a phenomenon that was, however, not observed for sterols. Analysis of subgroups and individual fatty acids, resin acids, and sterols identified using GC-MS revealed complex patterns, in which many, but not all, substances exhibited lower values in BSF and SBB materials than in REF materials. Overall, the results point towards the possibility to utilize a larger portion of wood logs from trees infested by beetles and fungi for value-added applications, such as pulping, rather than as fuel wood.

Lignin, the most abundant aromatic biopolymer in nature, is composed of phenylpropane units and represents a promising renewable source of aromatic chemicals for industrial applications. The valorization of lignin into bio-based chemicals through electrolyzers and upgrading technologies holds significant potential for developing environmentally and economically sustainable biorefineries. This minireview explores electrochemical hydrogen production coupled with alternative oxidation reactions that can replace the oxygen evolution reaction (OER), alongside discussions of lignin's structure, solubility, analytical methods, and the challenges of electrochemical depolymerization. Among various strategies, the electrocatalytic oxidation of lignin-derived phenolics has emerged as an environmentally benign approach, utilizing renewable electricity to drive reactions under mild and controlled conditions. Key topics include the development of efficient electrocatalysts for phenolic conversion and lignin-assisted proton exchange membrane electrolysis. Emphasis is placed on achieving high electrocatalyst activity, stability, and selectivity for effective lignin oxidation. Furthermore, challenges related to catalyst design, electrode materials, electrocatalytic systems, and process optimization are critically examined, along with potential pathways for improvement. This minireview highlights the opportunities and challenges in advancing electrocatalytic lignin valorization and provides perspectives on future developments in catalyst design and proton exchange membrane electrolysis integration to promote sustainable biomass utilization in accordance with green chemistry principles.

Developing effective fractionation methods remains challenging in biorefining. γ-Valerolactone (GVL) is a promising green solvent, yet its application in softwood biorefineries is still underexplored. In this study, GVL pretreatment for softwood biorefining was assessed at laboratory and pilot scales. The effects of temperature (170–210 °C), time (20–60 min), and GVL-to-water ratios (20:80 – 80:20 %) on the biorefining of spruce sawdust were initially investigated through lab-scale experiments using a 1-L reactor. A GVL/water solution at a 40:60 ratio, assisted by 0.4 g of sulfuric acid per 100 g of biomass, enabled the solubilization of up to 81.4 % of lignin and nearly the entire hemicellulosic fraction, while effectively preserving cellulose, which was subsequently saccharified with over 90 % conversion. Lignin was regenerated from the liquors and characterized using pyrolysis-gas chromatography/mass spectrometry, high-performance size-exclusion chromatography, Fourier-transform infrared spectroscopy, and 1H-13C heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy. The biorefinery concept was successfully scaled up and validated at pilot scale in a 50-L reactor, where enzymatic saccharification of the resulting cellulosic pulp produced hydrolysates that, upon fermentation, yielded 231.4 g of ethanol per kilogram of pulp. Lignin regenerated from the pilot-scale pretreatment liquors (118.9 g per kilogram of raw sawdust) and the lignin-rich saccharification residue (182.7 g/kg) were subjected to hydrothermal liquefaction, and the resulting biocrudes were characterized to assess their potential for biofuel formulation. The study showed the suitability of GVL for spruce biorefining to achieve high recovery of digestible cellulose, lignin and hemicelluloses fractions, that are subsequently valuable for chemicals and fuels production.

Background: Aeration plays a critical role in the bioconversion of pretreated lignocellulose by enhancing lytic-polysaccharide-monooxygenase(LPMO)-supported enzymatic saccharification. However, its broader impact, particularly on fermentation inhibitors, remains insufficiently understood. The hypothesis that aeration not only promotes LPMO activity, which has been shown clearly in previous studies, but also affects fermentation inhibitors was investigated in experiments with softwood pretreatment liquids. The effects of aeration were explored through chemical analysis of fermentation inhibitors and through subsequent fermentations with the xylose-utilizing Saccharomyces cerevisiae yeast CelluX^™4 to test the fermentability. Controls in which N₂ rather than air was supplied to the pretreatment liquids were used to distinguish between evaporation effects and effects caused by oxidation due to O₂ in air. In separate experiments, two redox-dependent detoxification methods, treatments with sulfite and laccase, were further investigated.

Results: While aeration had no negative effects on the subsequent fermentation of a sugar control, it compromised the fermentability of the pretreatment liquids. Compared to the N₂ control, subsequent fermentation of aerated samples showed reduced consumption of fermentable sugar (glucose, mannose, xylose) at 0.61 compared to 0.76 g L⁻¹ h⁻¹, and lower ethanol productivity (0.23 vs. 0.30 g L⁻¹ h⁻¹). Apart from more commonly studied pretreatment by-products (such as aliphatic carboxylic acids, furan aldehydes, and phenolics), methanol (~ 1 g L⁻¹) was detected in both pretreatment liquids. The methanol concentration decreased during gas addition, which was attributed to evaporation. Compared to the initial pretreatment liquid, aerated reaction mixtures exhibited slightly elevated levels of formaldehyde, but lower levels of furfural and vanillin. Sulfite detoxification was successful under both aeration and N₂ conditions. Treatment with laccase was found to have variable effects on the fermentability depending on the conditions applied.

Conclusions: The results underscore the dual role of aeration in softwood bioconversion, positive for promoting LPMO activity but potentially negative with respect to subsequent fermentability, and highlight the need to carefully tailor aeration strategies for the design of efficient biochemical processing of lignocellulosic feedstocks. Treatment with reducing agents, such as sulfite, emerges as a possibility to alleviate negative side-effects on the fermentability when aeration is used to promote LPMO activity.

Wood is the most abundant renewable natural resource composed of different polysaccharides and lignin, but its utilisation is hampered by intermolecular linkages between these components forming lignin-carbohydrate complexes (LCCs) causing recalcitrance. The links between glucuronoxylan and the γ-C of lignin (γ-ester linkages) are thought to contribute to one-third of LCCs, but direct evidence for their natural occurrence and their role in recalcitrance has been scarce so far. To address these issues, Phanerochaete carnosa glucuronoyl esterase (PcGCE), hydrolysing γ-ester linkages, was expressed in cell walls of developing wood in hybrid aspen (Populus tremula L. × tremuloides Michx.). The enzyme reduced HSQC 2D NMR signals corresponding to the γ-esters and xylan in dioxane-extracted LCCs without altering glucuronoxylan content or structure. This increased acid solubility of lignin and lignin content. Reduced wood recalcitrance was shown by increased sugar yields and glucose production rates (by approx. 20%) in saccharification without pretreatment and increased xylan extractability by subcritical water (by approx. 70%). Moreover, trees expressing PcGCE exhibited greater primary and secondary growth. Transcriptomics and metabolomics analyses in developing wood suggested that growth could have been induced by a higher transcription of SMR2 and RPOTmp, which was likely triggered by the secondary cell wall integrity signalling. The results provide evidence for the natural existence of LCC γ-esters and their significant contribution to lignocellulose recalcitrance. Furthermore, they show that reducing γ-ester linkages could increase plant productivity.

Preheating with hot air at 85 – 125 °C was evaluated for its effectiveness in removing terpenes and terpenoids in softwood sawdust, thereby enhancing fungal preprocessing and subsequent saccharification of softwood-based mushroom substrates. Sawdust from pine (Pinus sylvestris L.) and spruce (Picea abies (L.) H. Karst.) was preheated prior to shiitake (Lentinula edodes (Berk.) Pegler) cultivation. Preheating removed up to 96 % of terpenes in pine- based substrates and up to 50 % in spruce-based substrates. Additionally, preheating decreased total terpenoids content in spruce by up to 78 %. For the pine-based substrate, the mild heating generally led to faster colonisation and improved mushroom yield, with the fastest mycelia colonisation and highest yield observed for 105 °C treatment. This temperature was associated with the lowest content of total terpenes and absence of major monoterpenes. The content of terpenes and terpenoids continued to decrease during cultivation, alongside fungal degradation of lignocellulose. As a result of more extensive lignin degradation, the enzymatic digestibility of cellulose was higher for spruce-based spent mushroom substrate than for pine-based one (up to 89 % vs. 49 % conversion). Enzymatic digestibility showed a negative correlation with the α-pinene content, and a positive correlation with increasing preheating temperatures.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

Microbial exopolysaccharides (EPSs) have attracted increasing attention due to their versatile applications across diverse areas. However, large-scale production of EPSs remains challenging due to the high production costs, primarily driven by the use of synthetic carbon sources. This study demonstrates the potential of quinoa stalk hydrolysates as a sustainable alternative for EPS production using a halotolerant bacterial strain that was isolated from a hypersaline environment and termed SU4M. The bacterial isolate was identified through 16S rRNA and gyrB sequencing as a Bacillus swezeyi strain, and was then cultivated in quinoa stalk hydrolysates. The hydrolysates were produced by acid-catalyzed hydrothermal pretreatment using either sulfuric acid or phosphoric acid, followed by enzymatic saccharification. Fermentation experiments conducted in both shake flasks and bioreactors demonstrated that B. swezeyi SU4M utilized glucose from the hydrolysates efficiently, resulting in significantly higher biomass (5.1 ± 0.1 g L−1) and EPS production (1.2 ± <0.1 g L−1) compared to synthetic media (4.3 ± 0.1 g L−1 and 1.1 ± <0.1 g L−1). The kinetic analysis revealed distinct substrate consumption rates and growth patterns, with hydrolysates enhancing EPS yields under single-pulse fed-batch conditions. Advanced characterization techniques, including compositional analysis, Fourier transform infrared (FTIR) spectroscopy, 1H and 1H-13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR), high-performance size-exclusion chromatography (HPSEC), and thermogravimetric analysis (TGA), confirmed that the EPSs derived from hydrolysates were heteropolysaccharides with close structural similarities to those obtained from synthetic media. These findings underscore the potential of quinoa stalk hydrolysates as a biobased alternative to synthetic media as a substrate for EPS production.

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10 m³ bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48 h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.