Role of fermentation pH in enhancing hydrogen production from orange peel using crude enzyme catalysis

Dark fermentation is a promising strategy for biohydrogen production, but its low yield limits practical application. Crude enzyme extracted from orange peel has been reported to enhance hydrogen production rather than promote substrate hydrolysis during dark fermentation. Accordingly, this batch study investigated the influence of five initial fermentation pH levels (ranging from 5.5 to 9.5) on hydrogen production from synthetic swine wastewater via orange peel crude enzyme (OPCE)-mediated dark fermentation. The highest hydrogen yield (280.56 ± 5.34 mL/g glucose-added) was achieved at an initial pH of 6.5, where substrate conversion was most efficient and competing non-hydrogen pathways were minimized. Sludge at this pH exhibited a more favourable extracellular polymeric substance composition and structure, contributing to enhanced hydrogen production. A diverse microbial community was observed, with Firmicutes as the dominant phylum and Clostridium saccharoperbutylacetonicum, Clostridium sp., and Clostridium butyricum as key species, whose relative abundance varied with pH. These results highlight the critical influence of initial fermentation pH on optimizing OPCE-mediated dark fermentation for improved hydrogen yield.