Dual-matrix immobilization of cellulolytic consortium KKU-MC1 for Enhancing biohydrogen production from Napier grass and oil palm frond

Agricultural residues offer sustainable feedstock for biohydrogen production, but conventional processes face challenges including enzyme inhibition, poor cell retention, and limited catalyst reusability. This study compared separate hydrolysis and fermentation (SHF) versus simultaneous saccharification and fermentation (SSF) while developing a dual-matrix entrapment (ENT) system combining sodium-alginate with activated carbon. Using Napier grass (NG) and oil palm frond (10-50 g-volatile solid (VS)/L) without pretreatment, SHF outperformed SSF by 1.5-4.8 times across all conditions. Maximum hydrogen yield (HY) (119.9 ± 10.5 mL-H₂/g-VS) was achieved with the dual-matrix ENT system using NG at 10 g-VS/L. Scanning electron microscopy revealed hierarchical porous structures that enhanced enzyme diffusion and protected microbial cells. The dual-matrix ENT system maintained significantly higher cell concentrations and enzyme activity (r = 0.949 correlation with HY) through four reuse cycles, retaining 83.8 % of initial performance compared to 55.6 % for encapsulation (ENC) and 33.3 % for free cells. This approach creates ideal microenvironments for cellulolytic consortia while avoiding sugar inhibition, offering a practical solution for sustainable bioenergy production without energy-intensive pretreatment.