In Situ Formation of HAc/Ac^- Buffer Pair via Sodium Acetate Addition Enhances Two-Stage Continuous Biohythane...

Biohythane has attracted growing interest as an eco-friendly biofuel. However, its production stability is often compromised by rapid acidification during the dark fermentation hydrogen production stage. Acidification intensifies proton competition between ATP synthesis and H₂ evolution, leading to electron transfer inefficiency and metabolic disruption. While buffering systems can stabilize pH, the mechanisms by which adaptive buffers regulate microbial metabolism in two-stage biohythane fermentation remain unclear. In this study, sodium acetate (NaAc) was supplemented to a xylose-fed two-stage fermentation process, where it formed an in situ HAc/Ac^- buffer pair with the acetic acid generated endogenously during fermentation, achieving efficient continuous biohythane (H₂ + CH₄) production. The optimized semi-continuous system attained complete xylose degradation, with a maximum biohythane yield of 10.99 mol/mol, comprising 21.1% H₂ and 78.9% CH₄, representing an approximately 60% increase over the control. Volatile fatty acid and pH profiles indicated that NaAc addition provided a critical physicochemical environment that ensured high operational efficiency. Microbial analysis revealed that Caproiciproducens (52.08%) and Alcaligenes (16.94%) dominated the H₂ production stage, while Methanoculleus thermophilus (46.66%) and Methanothermobacter thermautotrophicus (37.94%) prevailed in the CH₄ production stage. This study proposes a feasible process control strategy for the two-stage biohythane fermentation process, which not only mitigates the acidification challenge in biological H₂ production but also enhances substrate-to-bioenergy conversion efficiency.