The production of H2 was studied using a packed bed reactor with polyurethane foam acting as support material. Experiments were performed using mixed microflora under non sterile conditions. The system was initially operated with synthetic wastewater as the sole substrate. Subsequently, cheese whey permeate was added to the system at varying organic loading rates (OLR). The performance of the reactor was evaluated by applying a continuous decrease in OLR. As a result, a significant decrease in H2 yields (HY) was observed with the decrease in OLR from 18.8 to 6.3 g chemical oxygen demand (COD)/L d. Microbial analysis demonstrated that the prevalence of non-hydrogen producers, Sporolactobacillus sp. and Prevotella, was the main reason for low HYs obtained. This behavior indicates that the fermentation under non-sterile conditions was favored by high concentrations of substrate by creating an adverse environment for nonhydrogen producer organisms.