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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.

Spent mushroom substrate (SMS), a byproduct of mushroom cultivation, has gained increasing attention as a sustainable soil amendment in agricultural and horticultural systems. SMS provides a promising alternative to peat-based substrates and synthetic fertilizers, supporting circular bioeconomy principles and sustainable farming practices. This is particularly relevant in boreal regions, where soils are typically acidic, nutrient-poor, and subject to short growing season conditions, presenting both opportunities and challenges for organic inputs. This review synthesizes current research on the agronomic and environmental implications of SMS use in agriculture and horticulture, including its effects on seed germination, plant growth, crop yield, root development, soil structure, and disease suppression. From an environmental perspective, the use of SMS contributes to waste reduction by repurposing organic residues and replacing peat, a non-renewable resource with significant ecological costs. However, several hurdles remain, including high salinity, inconsistent composition, nutrient imbalances, and complex compounds difficult for plants to access, which can impede plant performance. While prior studies have explored SMS in isolated settings, a comprehensive evaluation across systems is lacking. This review addresses that gap by assessing current evidence, identifying limitations, and outlining future research needs to optimize the use of SMS and scale its adoption in sustainable farming.

Supercritical carbon dioxide (scCO2) extraction offers environmental advantages over other techniques for extracting bioactive compounds from plant biomass. The effectiveness of scCO2 extraction can be enhanced by including a cosolvent in the process. When a vegetal oil is used as a cosolvent, further separation stages would be avoided, and a final product with improved functional and biological properties for food and nutraceutical products can be obtained. Coextraction or the joint extraction of bioactives from a primary solid matrix and an oil, acting as a cosolvent, from a secondary matrix, has been proposed as a solvent-free approach. This review presents an overview on the potential of coextraction as a strategy to valorize food and agricultural wastes and to develop novel products, containing both the solute and the cosolvent in a valuable, more stable, and bioavailable formulation of the bioactives.

This paper critically reviews and discusses the utilization of ultrasound as an eco-friendly approach for enhancing biomass valorization efficiency. In contrast to other methods, this process requires no additional chemicals, no post-treatment (such as wastewater treatment), and helps achieve industrial electrification goals. Ultrasound irradiation has been employed as a powerful tool in biomass pretreatment and biorefining, as well as a green extraction method for recovering bioactive molecules. By partially degrading lignin structures, ultrasound irradiation significantly improves the biological conversion of waste materials and lignocellulose, facilitating the exposure of valuable macromolecules, i.e., cellulose and hemicelluloses. This research begins with an introduction to sonication technology, and subsequently presents comprehensive discussions focusing on the application of ultrasound in the pretreatment of sludge and lignocellulosic materials for anaerobic digestion. Furthermore, various facets of ultrasound usage in bioethanol production, including substrate pretreatment, enzymatic and acid hydrolyzes, and fermentation techniques, are also examined. Additionally, the benefits of employing ultrasound technology to recover high-value, heat-sensitive bioactive compounds at temperatures below their degrading points, thereby preserving their functionality, are explored. Looking ahead, this review explores current trends in ultrasound technology adoption and its potential for scaling up and commercialization, introducing pathways for a more sustainable and efficient approach for biomass valorization.

Spent mushroom substrate (SMS), the main by-product of mushroom cultivation, is a source of sugars that can be released by saccharification. This work aimed at investigating the enzymatic saccharification of the polysaccharides of the SMS of shiitake (Lentinula edodes) and oyster mushroom (Pleurotus ostreatus) and exploring the lignin extraction from the saccharification residues. First, analytical enzymatic saccharification (AES) with a cellulase cocktail and an experimental hemicellulase-rich preparation was applied. AES revealed higher digestibility of both polysaccharides for shiitake SMS than for oyster mushroom SMS. Using the cellulase cocktail, shiitake SMS resulted in a digestibility above 80% and 70% (w/w) for cellulose and xylan, respectively, while the maximum values for oyster mushroom SMS were 52% and 32% (w/w). The experimental enzyme preparation resulted in lower cellulose digestibility and higher xylan digestibility. Still, the saccharification trend between the two SMS types remained unchanged. To enhance the enzymatic saccharification of oyster mushroom SMS, hydrothermal treatment was applied. The treatment improved the enzymatic digestibility of cellulose by up to 84%. A validation experiment at larger scale showed that hydrothermally treated oyster mushroom SMS had a comparable overall conversion with non-treated shiitake SMS. Following a biorefinery strategy, lignin was extracted from the residues of the preparative enzymatic saccharification using the green solvent γ-valerolactone under different temperatures and holding times. The extracted product contained 98.8% lignin and did not contain cellulose or xylan. The results of this study provide the grounds for biorefinery processes enabling recovery of bioactive compounds, fermentable sugars, and high-quality lignin from SMS.

This study aimed to assess subcritical water extraction (SWE) and pressurized ethanol extraction (PEE) for recovering bioactive compounds from spent mushroom substrate (SMS) derived from shiitake and oyster mushrooms. The extraction of bioactive compounds can facilitate the valorisation of SMS, an increasingly important waste stream whose management and disposal pose challenges for both mushroom producers and the environment. A 32 experimental design, using three temperatures (125, 150, and 175 °C) and three holding times (0, 15, and 30 min) was used in SWE. PEE was run at a single temperature (175 °C) and two holding times (0 and 15 min). The experimental results revealed that the highest extraction yield was achieved in shiitake SMS using SWE at 175 °C and 15 min. In most SWE conditions, shiitake SMS yielded higher concentrations of total phenolic compounds, total carbohydrates, and β-glucans compared to oyster mushroom SMS. Ascorbic acid, vanillic acid, protocatechuic acid, and gallic acid were the most abundant phenolic acids identified in both extracts. The SWE extracts of shiitake SMS exhibited higher DPPH and FRAP antioxidant activity than those of oyster mushroom SMS, and this activity increased with increasing temperature and time. PEE extracts exhibited higher DPPH and TEAC antioxidant activity for oyster mushroom SMS compared to shiitake SMS and showed a negative trend with increasing extraction time at 175 °C for both SMS. Our results show that SWE and PEE are viable methods for extracting bioactive compounds from SMS.

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.

Spent mushroom substrate (SMS), the residual material left after mushroom cultivation, represents an abundant yet underutilized bioresource. With global mushroom production generating millions of tons of SMS annually, its disposal constitutes a missed opportunity within the circular bioeconomy. This Opinion article highlights why SMS should be repositioned as a valuable raw material for sustainable biorefineries and outlines the technological, economic, and regulatory steps needed to unlock its potential.

A two-stage pretreatment toward lignocellulosic biomass fractionation was devised. The process consisted of dilute acid hydrolysis using oxalic acid, followed by oxalic acid–assisted ethanol organosolv pretreatment. A biomass mixture consisting of four regional lignocellulosic materials, namely, brewer’s spent grain, tomato waste biomass, cucumber waste biomass, and spent coffee grounds, was used. In the first stage, the optimum mixture composition was determined using a full factorial design coupled with a simplex-centroid design. The interactive effects of the solid-to-liquid ratio, holding time, acid type, and concentration were also considered. In the attempt to lower solid yields and increase hemicellulose dissolution, elevated levels of furfural (15.73 g/L) and 5-hydroxymethylfurfural (8.56 g/L) were formed, due to increased pretreatment severity (180 min, 135C, 83.15 mg oxalic acid/g biomass, and 100 g biomass/L). The solid yield achieved was 50.53%. In the second stage, the effect of ethanol-to-water solvent ratio, holding time, and temperature were investigated using a central composite experimental design. Solid yields ranged between 72.57 and 85.20% (w/w), mainly due to lignin removal. Pretreatment with 75% (v/v) ethanol at 120 min and 190 °C resulted in the highest lignin recovery (44.69%). Post-experimental verification runs were performed to evaluate the validity of the response surface models with a maximum error of 15.17%. Characterization by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and heteronuclear single quantum coherence spectroscopy (HSQC) were conducted to assess biomass fraction integrity and structural changes during pretreatment.

Shiitake mushroom cultivation could effectively reduce the recalcitrance of hardwood, facilitating cellulosic ethanol production. However, the shiitake pretreated spent mushroom substrates (SMS) have exhibited significant variations in enzymatic saccharification and fermentation efficiencies in previous studies. Parallel with shiitake cultivation, significant increases in extractives mass were observed in the SMS. This highlights the need to examine the formation of pretreatment by-products and their potential impact on downstream bioconversion processes. In this study, shiitake cultivation resulted in 42.6–47.6 % degradation of lignocellulosic components, with continuous generation of various small molecules. Shiitake demonstrated extensive and selective utilization of these degradation products during fructification. Consequently, non-utilized molecules, including (L)-dehydroascorbic acid, triglochinic acid, and 5-hydroxy-2-methylchromone, accumulated in SMS extractives as pretreatment by-products. The SMS cellulose showed 58.8 % digestibility upon enzymatic saccharification. The resulting hydrolysate was fermented to ethanol by Saccharomyces cerevisiae rendering 83.4 % of the theoretical yield. Although water extraction effectively minimized the by-products accumulation in SMS and hydrolysate, no improvement of enzymatic saccharification and fermentation efficiencies were observed. In contrast to thermochemical pretreatment methods, the by-products derived from shiitake pretreatment had limited inhibitory effects on downstream bioconversion processes. This study provides valuable indications for further optimization of shiitake pretreatment towards industrial implementation.