B. Zakaria, J. Panich, R. Swart, V.S. Nogue, C. Johnson, E. Sundstrom
Lawrence Berkeley National Laboratory, US Department of Energy,
United States
Keywords: biomanufacturing, formate, Cupriavidus necator, pH-stat bioreactors, hydrogen supplementation
Summary:
The urgent need to decarbonize our economy has heightened interest in carbon capture and utilization technologies. One promising avenue is the electrochemical reduction of CO2 to formic acid, a soluble C1 molecule that serves as both a carbon and energy storage medium. Formate serves as a versatile substrate for microbial growth and bioprocess applications, offering a promising avenue for sustainable biomanufacturing. Cupriavidus necator H16, a versatile soil bacterium capable of using formic acid as its sole carbon source, is particularly well-suited to upgrade CO2-derived formic acid into value-added chemicals, including sustainable aviation fuels. To further optimize its performance, we employed adaptive laboratory evolution (ALE) in continuous pH-stat bioreactors to enhance microbial fitness for formate utilization. However, the toxicity threshold of formate remains a bottleneck in scaling the process. Hydrogen offers an alternative energy source to complement formate, supporting its use as a carbon donor and alleviating toxicity challenges. Therefore, this study demonstrates the potential of coupling hydrogen with formate fermentation to enhance microbial growth and substrate utilization, offering a scalable approach for bio-based production systems. We first investigated formate fermentation using C. necator in fed-batch bioreactors operated under pH-stat mode. The pH was tightly regulated using a mixture of formic acid and ammonia, maintaining optimal growth conditions. The system operated by monitoring pH changes, where the consumption of formic acid increases the pH. This triggers the addition of formic acid to maintain a stable pH of 6.7, ensuring that formic acid is supplied at the same rate it is consumed. The formate concentration in the reactor was consistently maintained between 25–40 mM to avoid exceeding the critical toxicity threshold of formate on C. necator. Starting with an initial formate concentration of 20 mM, we achieved a significant substrate consumption, reducing formate from 5990 mM to 8.5 mM in 2.5 days at a substrate uptake rate of 173 mmol/L/h. This process achieved a high biomass, reaching an optical density (OD600nm) of 38. Then, we explored the impact of hydrogen addition on the fermentation process, providing extra reducing power and improving carbon and energy efficiency. Hydrogen also supported C. necator growth by acting as an energy source for carbon fixation pathways, while formate serves as the carbon donors, which mitigate formate toxicity, achieving high growth rates and biomass production. The supplementation of hydrogen demonstrated an enhanced rate of formate utilization. These findings highlight the potential of coupling formate fermentation with hydrogen supplementation to achieve higher substrate conversion rates and biomass yields. This integrated approach not only advances microbial biomanufacturing but also provides a pathway to efficiently upgrade CO2-derived formic acid into sustainable, value-added products.