Late-window oxygen pulsing unlocks lignin-carbohydrate shielding and enhances fiber deconstruction during maize...

Staged aeration offers a controllable lever to relax the strict-anaerobic paradigm in lignocellulosic fermentation. We compared single-dose air injection at early, mid, and late windows with a cellulase benchmark and anaerobic control, integrating fiber fractions, fermentation products, FTIR metrics, metagenomic functional profiles (CAZy and nitrogen cycling), microbial succession, and network topology. Mid and late aeration promoted ADL reduction and fiber deconstruction, lowering acid detergent lignin and acid detergent fiber by 23-34% and 13-15% versus the control, reaching cellulase-comparable levels. Late aeration produced the strongest structural unlocking, with attenuation of carbohydrate- and lignin-aromatic FTIR regions and reduced carbonyl-associated bands, consistent with disrupted lignin shielding and improved substrate accessibility. Late aeration increased glycosyltransferases yet showed the lowest glycoside hydrolases and auxiliary activities while achieving the largest net ADL and fiber losses, indicating that accessibility rather than terminal hydrolytic potential governs deconstruction intensity. Network analysis showed reduced mean degree and K-core but higher modularity under late aeration, consistent with a more compartmentalized interaction structure and reallocated carbon use. Overall, aeration timing is a scalable, low-input lever for process design. Future work should test generality across feedstocks and develop accessibility-based monitoring and control.