The hydrogen production by anaerobic hydrolysis can be a promising pathway to build up a carbon neutral hydrogen production. However, due to several factors, the potential faces several limitations. One important challenge is to reach the theoretical maximum hydrogen yield of 4 mole H-2 per mole glucose. This depends on the fermentative pathways which are influenced by different key parameters like the hydraulic retention time, organic loading rate, pH-value, volatile fatty acid production and the hydrogen partial pressure. The purpose was to evaluate the influence of certain parameters to find feasible strategies to reach high hydrogen yields by fermentation. For this reason, two-phase fermentation trials were carried out and monitored.