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 O-2 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 O-2), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O-2. 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 O-2 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 O-2 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. (C) 2003 Wiley Periodicals, Inc.