Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of ${\text{CO}}_2$ and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during $H_2$ -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of $H_2$ and ${\text{CO}}_2$ when supplemented with nitrate. Transcriptomic analysis, ${13}^{}{\text{CO}}_2$ labeling, and an electron balance were used to understand how electron flux was partitioned between ${\text{CO}}_2$ and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the ${\text{CO}}_2$ fixation rate and the production metrics of other chemicals from ${\text{CO}}_2$ and $H_2$ in this host.