H₂ drives metabolic rearrangements in gas-fermenting Clostridium autoethanogenum

The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H₂ should diminish substrate loss to CO₂ and facilitate production of reduced and energy-intensive products. However, the effects of H₂ supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions.

Here, we quantify the effects of H₂ supplementation by comparing growth on CO, syngas, and a high-H₂ CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model. CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H₂. We show that H₂ supply strongly impacts carbon distribution with a fourfold reduction in substrate loss as CO₂ (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H₂ supplementation lowers the molar acetate/ethanol ratio by fivefold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H₂ availability provided reducing power via H₂ oxidation and saved redox as cells reduced all the CO₂ to formate directly using H₂ in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity.

Our quantitative molecular analysis revealed that H₂ drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H₂ supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.