The effect of substrate concentrations (0, 7.5, 15, 22.5, 30, and 37.5 g-VS/L) on hydrogen production from heat-treated and fresh food waste (FW) using untreated inoculums was investigated in this work. The highest hydrogen yield (75.3 mL/g-VS) was obtained with heat-treated FW at 15 g-VS/L. Lower substrate content could not provide enough organic matter for hydrogen fermentation, while higher substrate concentrations shifted the metabolic pathways from hydrogen fermentation to lactic acid fermentation by enriching the lactic acid bacteria (LAB), which lowered the slurry pH and decreased enzyme activity, resulting in a lower chemical oxygen demand (COD), volatile solid (VS), carbohydrate removal rate, and hydrogen yield. Compared with fresh FW, heat-treated FW is preferred for biohydrogen process with acetate as the main organic product. Additionally, at the optimal concentration (15 g-VS/L) using fresh FW, lactic acid is first accumulated and then degraded to produce hydrogen with butyrate as the main metabolite. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.