Gas fermentation represents a promising strategy to recycle emitted carbon into value-added products, thereby reducing dependence on fossil resources. The chemolithoautotrophic bacterium Cupriavidus necator enables the conversion of CO₂-rich, oxygen-containing gas streams into chemicals such as isopropanol. This study established and characterised isopropanol formation as a model process for aerobic gas fermentation.A small-scale shake flask system was developed to enable reproducible autotrophic cultivations under overpressure up to 1.5 bar, enhancing gas transfer and supporting growth rates of 0.13–0.15 h⁻¹. Medium composition was adapted to autotrophic conditions, with a focus on nickel feeding. Eight engineered C. necator strains were evaluated under heterotrophic and autotrophic conditions. While growth rates were similar among strains, heterotrophic cultures grew faster (μ = 0.20–0.26 h⁻¹) and exhibited higher product formation rates, which were reduced by 46–62% under autotrophy. Under heterotrophy, inducible strains containing PBad and Prham promoters were among the best performing strains while showing low product formation in autotrophy under nitrogen depletion. In contrast, a strain harboring the nitrogen-responsive Pgln promoter maintained product formation under these conditions, demonstrating its suitability for autotrophic cultivation. Furthermore, co-expression of PHB synthesis decreased the isopropanol yield, confirming the benefit of using PHB-deletion mutants as production hosts.At bioreactor scale, controlled overpressure of up to 3 bar, multiple gas spargers, and in situ gas probes allowed a precise gas feed strategies. The set-up further allowed determining the gas uptake rates, providing a base for future economic evaluations. The PHB-deletion strain Re2133 achieved cell densities up to 23 g L⁻¹, and Re2133/pEG7c produced 11 g L⁻¹ isopropanol, which is the highest concentration reported for C. necator under autotrophic conditions. Finally, process robustness was confirmed with real industrial off-gases as carbon source, demonstrating comparable growth and product formation despite gas impurities.
Isabell Weickardt (Wed,) studied this question.