Hybrid PVA-alginate immobilization enables configuration-dependent biohydrogen production from Napier grass and oil palm fronds with superior multi-cycle stability

Dual-matrix approaches face stability limitations (83.8% retention, 4 cycles). We developed a simplified hybrid polyvinyl alcohol (PVA)-alginate immobilization system (4.50% PVA, 1.50% alginate, 0.25% silica, 0.50% chitosan) that independently immobilizes cellulolytic organisms (modified KKU-MC1) and hydrogen (H2)-producing bacteria (HPB). This platform enabled systematic evaluation of four process configurations combining simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) with selective immobilization using Napier grass (NG) and oil palm fronds (OPF). Configuration profoundly influenced performance: SHF1 (free modified KKU-MC1 + immobilized HPB) achieved 81.5 ± 7.5 mL-H2/g-volatile solid (VS) from NG 2.2-fold higher than SSF while SSF2 (immobilized modified KKU-MC1 + free HPB) demonstrated extraordinary co-digestion synergy at 7.5:2.5 NG:OPF ratio (61.6 ± 7.4 mL-H2/g-VS, 12.5-fold the theoretical). Five-cycle testing revealed 84.0–89.1% H2 retention, 50.0–66.7% enzyme retention, and 50.0–80.0% biomass retention with intact beads, outperforming dual-matrix systems (5 vs. 4 cycles). Process configuration and substrate characteristics proved the primary determinants of H2 yield, while medium components represented 57.3% of costs. With five-cycle stability providing 5.0-fold economic advantages (0.00364 USD-H2/USD-total), this platform establishes configuration-substrate-immobilization as critical for agricultural bio-H2 production.