The technical feasibility of Cupriavidus necator DSM 545 for sustainable autotrophic polyhydroxybutyrate (PHB) production from CO2 using a two-stage cultivation system was evaluated. In this cultivation method, cell mass growth occurred under heterotrophic conditions using two different organic substrates, namely glucose and waste glycerol. In both cases, PHB biosynthesis was triggered by applying nitrogen and oxygen limitation at three different cell mass concentrations under autotrophic conditions using a gas mixture of H-2, O-2 and CO2. To ensure that the test conditions were relevant for later industrial application, O-2 concentration was kept below the safety value during autotrophic PHB production. PHB production from CO2 on waste-glycerol grown cell mass resulted in a PHB concentration of 28 g/L, which is the highest reported value in literature for PHB synthesized from CO2 at an O-2 concentration below the lower explosion limit of 5 vol%. The fermentation performance decreased when nutrient limitation was delayed at higher cell mass concentrations. Furthermore, it was shown that PHB production from CO2 at high cell mass concentration is metabolically feasible, but under the tested conditions the mass transfer of O-2 was limiting PHB accumulation. Characterization of the produced polymers showed that the organic carbon source affected the properties of PHB and that the cultivation method developed in this study provided PHB with properties similar to a commercial PHB and PHB typically found in literature. It can be concluded that heterotrophic-autotrophic production of PHB by C necator is a promising cultivation method to reduce the overall production cost of PHB. In order to compete with the current heterotrophic cultivation system, the oxygen transfer rate must however be enhanced to achieve a higher PHB productivity. (C) 2014 Elsevier B.V. All rights reserved.