Comparative Secretome Analysis and Enzyme Cocktail Optimization of Six Fungal Species Under Solid-State and Submerged Fermentation for Lignocellulosic Saccharification of Flax Shives

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biorefinery applications, yet efficient enzymatic saccharification remains challenging due to the recalcitrant structure of plant cell walls. This study presents a comprehensive comparative analysis of enzymatic activities, saccharification performance, and secretome composition of six fungal species cultivated under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using untreated flax shives as substrate. While SmF yielded approximately 4-fold higher total protein concentrations (0.38 ± 0.13 g.L ^-1 vs. 0.08 ± 0.02 g.L ^-1 ), SSF-derived enzymes demonstrated superior specific enzymatic activities, particularly for endo-xylanase and endo-cellulase, resulting in more efficient biomass saccharification. Proteomics analysis revealed distinct secretome profiles between fermentation modes, with SSF showing higher proportions of polysaccharide metabolism proteins (71.0%) compared to SmF (49.3%), while SmF exhibited greater enzyme diversity including more lytic polysaccharide monooxygenases (LPMOs) and auxiliary activity enzymes. Trichoderma species consistently demonstrated the highest saccharification efficiency, with glucose yields reaching 2.37 mM under SSF conditions. A Scheffé simplex-lattice mixture design comprising 65 enzyme cocktail combinations revealed significant synergistic interactions between several cocktails, with the binary mixture of Trichoderma 2SA21 and P. chrysogenum achieving 54% synergy - in terms of higher sugar release above expectations - and the highest total monosaccharide release (1.80 mM). These findings provide practical guidance for developing cost-effective enzyme cocktails for lignocellulosic biorefinery applications, emphasizing the importance of fermentation mode selection and strategic strain combination over enzyme supplementation complexity. The methodology established here, combining systematic screening, comparative proteomics, and statistical mixture design, offers a robust framework for optimizing fungal enzyme systems across diverse biomass substrates.