Comparative characterisation of autotrophic and heterotrophic isopropanol formation by Cupriavidus necator in shake flasks

Autotrophic cultivation offers a path to carbon-neutral bioproduction, which is increasingly valuable in the context of climate change mitigation. In this study, the production of isopropanol by Cupriavidus necator is used as an example for CO₂ valorisation, and a simple shake bottle system is introduced to facilitate the development of aerobic autotrophic cultivation processes and strain screening. Applying 1.5 bar overpressure in the bottle's headspace enhances gas transfer while pressure decrease was shown to be correlated to biomass and product formation, allowing to follow metabolic activity without sampling. After optimizing cultivation parameters and nickel feeding strategy, the system was applied to compare three different isopropanol-producing strains. The highest autotrophically obtained isopropanol concentration was 2.2 ± 0.5 g L⁻¹ with a specific yield of 0.9 ± 0.2 g g_CDW⁻¹ and a minimal by-product concentration of 0.05 ± 0.01 g L⁻¹ acetone. Heterotrophic cultivations were carried out for comparison, obtaining up to 3.4 ± 0.2 g L⁻¹ final isopropanol concentration with a specific yield of 1.4 ± 0.1 g g_CDW⁻¹. Although the use of CO₂ instead of fructose resulted in a slower process, the overall isopropanol production is promising. This study provides valuable insights into strain behaviour while demonstrating the utility of the presented shake bottle system for advancing autotrophic process development.