ABSTRACTThis study conceptualizes the design of a small‐scale system (250 mL–1 L) for the autotrophic cultivation of hydrogen‐oxidizing bacteria, such as the representative strain Cupriavidus necator. The research aimed to systematically investigate the impact of bottle volume and gas composition, particularly oxygen concentration, on the growth and performance of C. necator during autotrophic cultivations. To this end, customized, pressure‐tight, baffled glass bottles of various sizes (250, 500, 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 were calculated. Among the various combinations, the 1000‐mL bottles demonstrated the highest µmax (0.13 h−1) and the second‐highest BPR (0.074 g L−1 h−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 minimize 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.