In this study, we evaluated the effectiveness of lake sediment as inoculum for hydrogen production through dark fermentation in a repeated batch process. In addition, we investigated the effect of heat treatment, applied to enrich hydrogen-producing bacteria, on the bacterial composition and metabolism. Denaturing gradient gel electrophoresis and molecular cloning, both performed using the 16S rDNA gene as target gene, were used to monitor the structure of the bacterial community. Hydrogen production and bacterial metabolism were analysed via gas chromatography and high-performance liquid chromatography. Both treated and non-treated inocula were able to produce high amounts of hydrogen. However, statistical analysis showed a clear difference in their bacterial composition and metabolism. The heat treatment favoured the growth of different Clostridia sp., in particular of Clostridium bifermentans, allowing the production of a constant amount of hydrogen over prolonged time. These cultures showed both butyrate and ethanol fermentation types. Absence of heat treatment allowed species belonging to the genera Bacillus, Sporolactobacillus and Massilia to outgrow Clostridia sp. with a reduction in hydrogen production and a significant metabolic change. Our data indicate that lake sediment harbours bacteria that can efficiently produce hydrogen over prolonged fermentation time. Moreover, we could show that the heat treatment stabilizes the bacterial community composition and the hydrogen production.