<p>Biohydrogen (BioH₂) production from waste resources, such as food waste, is a potential source of sustainable and clean energy. Previous literature has reported enhancement in the kinetics and yield of dark fermentation for bioH₂ production using sonication. However, the mechanism by which sonication affects the cellular metabolism has remained largely unexplored. The present study aims to investigate the effect of ultrasound on the metabolic network of <i>Clostridium pasteurianum</i> during the dark fermentation of food waste hydrolysate and to elucidate the underlying mechanism using metabolic flux analysis (MFA). A metabolic flux model was developed to determine the impact of sonication on intracellular metabolite fluxes. Hexose sugar uptake increased by ~ 47% with sonication, while butyrate and acetate fluxes at the acetyl-CoA node increased by ∼9% and ∼94%, respectively. Sonication improved bioH₂ yield by ∼22%, and the acetate-to-butyrate (A/B) ratio by ∼37%. These results pointed out that bioH₂ production is linked to carbon flux at the acetyl-CoA node. A higher flux towards the acetate route (compared to the butyrate route) enhances hydrogen yield. Based on these results, a hypothetical MFA analysis (with sonication) was conducted for two cases: (1) complete redirection of carbon flux at the acetyl-CoA node to the acetate route, and (2) doubling the uptake flux of hexose sugars. For the first case, bioH₂ enhanced from 4.13 to 6.47 mmol/L⋅h, while in second case, bioH₂ flux of 14.53 mmol/L⋅h was predicted by MFA model. These results could be useful for the genetic engineering of microbial strains for enhanced bioH₂ production.</p>