Project description:Background: 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. Results: 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 (GEM). 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 four-fold 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 five-fold. 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. Conclusions: 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.
Project description:Clostridium ljungdahlii not only utilizes CO, but also H2 as energy source during autotrophic growth. In theory, CO is a more energetically and thermodynamically favourable energy source than H2 in the gas fermentation of C. ljungdahlii. However, how C. ljungdahlii conserves energy for growth and ethanol/acetate formation grown on CO or CO2/H2 is not in great detail. In this study, C. ljungdahlii was fermented on CO and CO2/ H2 at pH 6.0 with 0.1 MPa gas pressure. C. ljungdahlii produced 27 g/L acetate, 9 g/L ethanol, 8 g/L 2,3-butanediol and traces of lactate in the presence of CO as energy source, while it produced 25.8 0.1 g/L acetate, 1.8 0.1 g/L ethanol, 0.7 0.01g/L 2,3-butanediol and trances of lactate in the same fermentation condition using H2 as energy source. Therefore, comparative transcriptomes between cells grown on CO and cells grown on H2/CO2 were performed to investigate gene expression profiles based on three biological replicates.