Conceptual Approach for Aerobic Autotrophic Gas Cultivation in Shake Flasks: Overcoming the Inhibitory Effects of Oxygen in Cupriavidus necator

This study conceptualises the design of a small-scale system (250mL-1L) for the autotrophic cultivation of the hydrogen-oxidising bacteria, such as representative strain Cupriavidus necator. The research aimed to systematically investigate the impact of the bottle volume and gas composition, particularly oxygen concentration, on the growth and performance of C. necator during autotrophic cultivations. To this end, customised pressure-tight, baffled glass bottles of various sizes (250 mL, 500 mL, and 1000 mL) and gas mixtures with varying oxygen concentrations (4%, 8%, and 12% v/v) were tested. Growth was monitored by measuring optical density. The maximum specific growth rate (μmax), the biomass production rate (BPR), the volumetric gas-liquid mass transfer coefficient (kLa), and the oxygen transfer rate (OTR) were calculated. Among the various combinations, the 1000 mL bottles demonstrated the highest μmax (0.13 h-1) and second-highest BPR (0.074 gL-1h-1) at an oxygen concentration of 8%, without the need to refill the headspace.

The proposed small-scale system offers a swift and replicable method for concurrently investigating multiple autotrophic cultivations. In this regard, increasing the size of the bottle flask proved to be an efficient strategy to minimise the periodicity for gas refilling. Due to the inhibitory effect of oxygen, changing the liquid-gas volume ratio in hydrogen-driven shake flask cultivation had so far strongly influenced the growth rate. Our results provide a solid foundation for the scaling and optimization of small-scale cultivation of chemolithotrophic bacteria and will facilitate future parallelization and hence optimization of metabolic aspects.