Minimizing the Lag Phase of Cupriavidus necator Growth under Autotrophic, Heterotrophic, and Mixotrophic Conditions

Cupriavidus necator has the unique metabolic capability to grow under heterotrophic, autotrophic, and mixotrophic conditions. In the current work, we examined the effect of growth conditions on the metabolic responses of C. necator. In our lab-scale experiments, autotrophic growth was rapid, with a short lag phase as the exponential growth stage was initiated in 6 to 12 h. The lag phase extended significantly (>22 h) at elevated O₂ and CO₂ partial pressures, while the duration of the lag phase was independent of the H₂ or N₂ partial pressure. Under heterotrophic conditions with acetate as the organic substrate, the lag phase length was short (<12 h), but it increased with increasing acetate concentrations. When glucose and glycerol were provided as the organic substrate, the lag phase was consistently long (>12 h) regardless of the examined substrate concentrations (up to 10.0 g/L). In the transition experiments, C. necator cells showed rapid transitions from autotrophic to heterotrophic growth in less than 12 h and vice versa. Our experimental results indicate that C. necator can rapidly grow with both autotrophic and heterotrophic substrates, while the lag time substantially increases with nonacetate organic substrates (e.g., glucose or glycerol), high acetate concentrations, and high O₂ and CO₂ partial pressures. <b>IMPORTANCE</b> The current work investigated the inhibition of organic and gaseous substrates on the microbial adaption of Cupriavidus necator under several metabolic conditions commonly employed for commercial polyhydroxyalkanoate production. We also proposed a two-stage cultivation system to minimize the lag time required to change over between the heterotrophic, autotrophic, and mixotrophic pathways.