Leveraging overpressure to improve gas conversion in Methylococcus capsulatus scale-up fermentations

Gas transfer limitations represent a major bottleneck in methanotrophic fermentations, restricting the industrial implementation of methane-to-biomass conversion processes. This study investigated the application of elevated pressure as a strategy to overcome gas transfer limitations during scale-up of Methylococcus capsulatus (Bath) cultivation from shake flasks to reactor scale. A systematic scale-up approach was employed, from 250 mL shake flasks through 1.5 L benchtop to a novel 7.5 L automated pressure stirred-tank reactor system with ATmospheres EXplosives (ATEX) certification. The pressure-controlled system enabled real-time dissolved oxygen tension (DOT)-controlled adjustment of headspace pressure, allowing independent optimization of gas transfer rates while maintaining process safety. Comparative experiments demonstrated successful scale-up with maintained oxygen transfer rate, methane transfer rate, and carbon dioxide transfer rate reproducibility across all scales. Gauge pressures up to 7 bar(g) significantly enhanced gas-liquid transfer, increasing gas utilization efficiency from 33% to 60% and biomass formation by 38% compared to atmospheric conditions. The DOT-controlled pressure strategy reduced oxygen limitation and maximized transfer rates. The developed pressure fermentation system successfully addressed key industrial scaleup challenges, including ATEX safety requirements, real-time process monitoring, and enhanced gas conversion efficiency. This approach demonstrates the potential of pressure enhanced bioprocessing for industrial methane valorization, providing a scalable platform for sustainable greenhouse gas conversion technologies.