In Situ Formation of HAc/Ac− Buffer Pair via Sodium Acetate Addition Enhances Two-Stage Continuous Biohythane Production from Xylose: Process Performance and Microbial Community Dynamics

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.