Quantifying microbial, enzymatic, and acid-driven pathways of lignocellulose degradation and their impact on methane production in Sudangrass silage

Abstract

Lignocellulosic biomass represents a promising feedstock for the sustainable circular bioeconomy. However, its recalcitrant structure necessitates efficient pretreatment. Converting lignocellulosic biomass into renewable energy via anaerobic digestion (AD) often exhibits low degradation efficiency and thus requires optimization to enhance conversion efficiency. Ensiling is a promising, low-cost, and environmentally friendly biological pretreatment method, yet the individual contributions and interactive mechanisms of microbial-, enzymatic-, and acid-driven pathways during lignocellulose degradation have not been fully elucidated. In this work, gamma-ray sterilization, high-temperature enzyme inactivation, and organic acid simulation were employed to isolate and quantify the effects of enzymatic-driven (ED), acid-driven (AD), microbial-driven (BD), and synergistic (SYN) pathways on lignocellulose degradation and methane production during Sudangrass ensiling. Results demonstrated that the BD treatment, driven by synergistic microbial activity and organic acids, achieved the highest lignocellulose degradation rate (LDR, 24.5%) and methane yield (295.20 mL/g VS). The absence of active microbiota in the AD and ED treatments significantly decreased LDR (13.6% and 4.36%, respectively) and reduced methane production (275.50 mL/g VS and 274.40 mL/g VS). The SYN treatment had moderate performance with LDR of 20.3% and methane yield of 284.20 mL/g VS. Structural equation modeling (SEqM) revealed that enzymatic and acid pathways affected methane yield only indirectly via water-soluble carbohydrate release, while the microbial pathway exerted dual direct and indirect effects by simultaneously promoting lignocellulose degradation and sugar metabolism. Furthermore, the potential synergy between microbes and endogenous enzymes was inhibited by rapid acidification. These findings confirm that microbial-driven processes are the dominant mechanism enhancing lignocellulose biodegradability and methane production. This work provides mechanistic insights for optimizing ensiling-based pretreatment to boost the efficiency of anaerobic digestion of lignocellulosic biomass.