Protocol for controlled gas-phase oxygen In-leakage in anaerobic bioreactors

Trace oxygen may enter the gas supply line of an anaerobic bioreactor and affect reactor operation. In this study, we describe a method to expose a defined oxygen concentration for gas-fed anaerobic bioreactors. H₂, CO₂ and premixed CO₂/O₂ gas were supplied using mass flow controllers, and the reactor inlet O₂ concentration was adjusted from 0 to 10,000 ppm. Both stepwise and constant exposure tests were carried out. The reactor outlet O₂ concentration was measured by gas chromatography to confirm the actual exposure condition in bioreactors. Because oxygen exposure is defined at the reactor inlet, stable exposure conditions can be applied regardless of oxygen consumption within the reactor. This protocol provides an approach for investigating trace oxygen responses in continuously operated anaerobic microbial cultures.•Defined inlet O2 concentrations can be generated by gas blending using mass flow controllers.•Oxygen exposure conditions can be monitored by outlet gas chromatography.•This method can be applied to stepwise and continuous oxygen exposure experiments.