Brewer’s spent grain (BSG), the main lignocellulosic by-product of the beer industry, represents an abundant yet underutilized resource with high potential for valorization. This study presents an integrated biorefinery approach to convert BSG into second-generation (2G) ethanol, bioactive vinasse for plant growth promotion, and fungal biomass as a potential mycoprotein source. The biomass was first subjected to biological delignification using the white-rot fungus Ganoderma lucidum, after which two valorization routes were explored: (i) evaluation of the fungal biomass as a mycoprotein candidate and (ii) alcoholic fermentation for ethanol production. For the latter, three pretreatment strategies were assessed (diluted sulfuric acid and two deep eutectic solvents (DESs) based on choline chloride combined with either glycerol or lactic acid) followed by a one-pot enzymatic saccharification and fermentation using Kluyveromyces marxianus SLP1. The highest ethanol yield on substrate (Y_P/S) was achieved with [Ch]Cl:lactic acid pretreatment (0.46 g/g, 89.32% of theoretical). Vinasse, recovered after distillation, was characterized for organic acid content and tested on Solanum lycopersicum seed germination, showing promising biostimulant activity. Overall, this work highlights the potential of BSG as a sustainable feedstock within circular economy models, enabling the production of multiple bio-based products from a single residue.

Brewer’s spent grain (BSG) is an abundant lignocellulosic by-product whose valorization can support circular bioeconomy strategies. This study evaluated BSG bioconversion by Aspergillus oryzae ATCC 10124 under solid-state fermentation (SSF) to produce lignocellulolytic enzymes and release second-generation (2G) sugars relevant to biorefinery applications. SSF was monitored over 0–10 days, and FPase, endo-cellulase, β-glucosidase, xylanase, mannanase, amylase, and ligninolytic enzyme activities were quantified. Enzymatic crude extracts were further assessed in SDS-PAGE analysis. Glucose, cellobiose, xylose and arabinose release and consumption were tracked throughout fermentation, and substrate transformation was supported by FTIR. The secretome exhibited a predominantly hydrolytic profile, with maximal hemicellulolytic and cellulolytic activity around days 2–4, as well as sustained amylase activity. Ligninolytic activity was not detected. Sugar profiles indicated rapid early hydrolysis of glucose, followed by progressive pentose release. The stabilization and decline were consistent with fungal uptake. Changes in the carbohydrate fingerprint and SDS–PAGE banding supported structural polysaccharide remodeling and hydrolytic protein secretion. Thus, this SSF platform confirmed certain potential for low-cost cellulolytic and hemicellulolytic enzyme generation. However, because sugar accumulation was temporary and followed by consumption, this system is best interpreted as a biological pretreatment and enzyme-generation step that supports subsequent downstream valorization.