One of the main disadvantages of the dark fermentation process is the cost associated with the stages needed for obtaining H₂ producing microorganisms. Using anaerobic microflora in fermentation systems directly is an alternative which is gaining special interest when considering the implementation of large-scale plants and the use of wastes as substrate material. The performance of two H₂ producing microflora obtained from different anaerobic cultures was studied in this paper. Inoculum obtained from a waste sludge digester and from a laboratory digester treating slaughterhouse wastes were used to start up H₂ fermentation systems. Inoculum acclimatized to slaughterhouse wastes gave better performance in terms of stability. However, due to the limited availability of this seed material, further work was performed to study the behaviour of the inoculum obtained from the municipal wastewater treatment plant. The process was evaluated under static and mixing conditions. It was found that application of a low organic loading rate favoured the performance of the fermentation systems, and that agitation of the reacting mass could alleviate unsteady performance. Specific H₂ production obtained was in the range of 19–26L/kg SV_fed with maximum peak production of 38–67L/kg SV_fed. Although the performance of the systems was unsteady, recovery could be achieved by suspending the feeding process and controlling the pH in the range of 5.0–5.5. Testing the recovery capacity of the systems under temperature shocks resulted in total stoppage of H₂ production.