Assessment of Different Strategies for Acetic Acid Production from Gaseous CO ₂ and N ₂ Feedstock Using <i>Clostridium ljungdahlii</i>

As carbon and nitrogen sourcing impacts both the economic and environmental aspects of bioprocesses, substituting sugar-derived carbon and ammonia-based nitrogen salts with renewable gaseous inputs offers a sustainable route for biomanufacturing. The bioproduction of chemicals using gaseous feedstocks such as CO2, N2, and H2 can therefore reduce carbon emissions while decreasing the dependence on fossil-based nutrient resources. Here, we investigate the capability of Clostridium ljungdahlii, a well-studied C1-fixing model anaerobic bacterium, to produce acetate solely from N2 and CO2 via gas fermentation and microbial electrosynthesis (MES) processes. It produced 77.32 ± 8.68 mg/L (7.73 ± 0.87 mg/L/day) and 50.34 ± 3.99 mg/L (5.59 ± 0.44 mg/L/day) acetate under optimized gas fermentation conditions (37 °C, 1 atm pressure, 1:4 N2:H2 ratio, 10% v/v (OD600 = 0.08) inoculum size) and MES experiments at an applied cathode potential of −1.2 V (vs Ag/AgCl), respectively. C. ljungdahlii evolved through an adaptive laboratory evolution strategy produced 115.98 ± 4.38 mg/L acetate at an improved rate (16.57 ± 0.63 mg/L/day) in gas fermentation experiments. Trace fructose (0.55 mM) addition to the minimal growth medium, determined by metabolic flux analysis, increased acetate production to 279.34 ± 10.01 mg/L at a 69.83 ± 2.51 mg/L/day rate. Control experiments and nitrogen mass balance indicated that l-cysteine mainly supports early culture growth as a reducing agent and a transient nitrogen donor, whereas the nitrogen required for sustained biomass growth could not be accounted for by media impurities or dead biomass recycling. A comparative numerical analysis considering cost, production rate, and carbon emissions suggests that trace fructose addition is a feasible strategy for bioproduction from gaseous substrates. This study presents a promising N2- and CO2-based biomanufacturing approach that circumvents the use of ammonia-derived fixed nitrogen with potential applications for utilizing industrial N2- and CO2-containing flue gases from thermal power plants and coal-fired boilers.