Dark fermentative H2 production is an important route to renewable energy as it is based on a known technology and can utilize a wide range of available waste streams as substrate. However, more research is required to overcome the technical barriers to practical application. The aim of this study is to investigate different scenarios towards the optimization of fermentative H2 production from synthetic and real wastes using pure and mixed cultures. Lignocellulosic biomass, i.e. pretreated corn cobs and poplar wood hydrolysate were evaluated for H2 production using mixed anaerobic cultures and yields of 141 and 169 mL H2/gCODadded were determined, respectively. Also, substrate utilization kinetic parameters for selected mesophilic and thermophilic H2-producing pure cultures utilizing hexose and pentose sugars were determined. Furthermore, the effect of co-fermentation and co-cultures on H2 production was studied. This work proved that headspace CO2 sequestration in a continuous-flow system producing H2 from glucose increased H2 yield from 2.4 to 3.0 mol/mol glucose, i.e. approximately 90% of the theoretical yield. An extensive comparative study of mesophilic and thermophilic anaerobic digester sludges confirmed the superiority of thermophilic cultures which produced 23.8 L H2/L poplar wood hydrolysate. The Monod kinetic parameters of mono- and co-culture of Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum on glucose, starch, and cellulose were derived