Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: Evaluation by CO₂ pulse injection and autogenous CO₂ methods

In order to see the effect of CO₂ inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O₂ were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO₂ concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O₂), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O₂. We used two different experimental methods for evaluating CO₂ inhibition: CO₂ pulse injection and autogenous CO₂ methods. A 10 or 22% (v/v) CO₂ pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO₂ affects the synthesis of biomass and PHB. CO₂ inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO₂ concentration and pulse duration. The new proposed autogenous CO₂ method makes it possible to place microbial cells under different CO₂ level environments by varying the gas flow rate. Introduction of O₂ gas at a low flow rate of 0.42 vvm resulted in an increase of CO₂ concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O₂ flow rate. This new method measures the inhibitory effect of CO₂ produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO₂ inhibition without introducing artificial CO₂ into the fermentor.