The buffering effect of acetate on hydrogen production during glucose fermentation by Ethanoligenens harbinense B49 was investigated compared to phosphate, a widely used fermentative hydrogen production buffer. Specific concentrations of sodium acetate or phosphate were added to batch cultures, and the effects on hydrogen production were comparatively analyzed using a modified Gompertz model. Adding 50 mM acetate or phosphate suppressed the hydrogen production peak and slightly extended the lag phase. However, the overall hydrogen yields were 113.5 and 108.5 mmol/L, respectively, and the final pH was effectively controlled. Acetate buffered against hydrogen production more effectively than did phosphate, promoting cell growth and preventing decreased pH. At buffer concentrations 100-250 mM, the maximum hydrogen production was barely suppressed, and the lag phase extended past 7 h. Therefore, although acetate inhibits hydrogen production, using acetate as a buffer (like phosphate) effectively prevented pH drops and increased substrate consumption, enhancing hydrogen production.