Exogenous CO2 Modulates Methane Production in Anaerobic Digestion: Insights into Substrate-Dependent Metabolic Regulation

Exogenous CO2 injection has shown inconsistent effects on anaerobic digestion performance due to variations in substrate properties and operational parameters. This study investigated the effects of CO2 injection on microbial community composition, key metabolic pathways, and methane yield. CO2 influenced bacterial populations, promoting the enrichment of hydrolytic acidogenic microbes that accelerate the breakdown of complex organics and generate precursors for methanogenesis, while methanogenic archaea remained stable, maintaining both acetoclastic and hydrogenotrophic pathways. Functional gene analysis showed enhanced abundances in the Acetyl CoA and Wood Ljungdahl pathways, indicating improved carbon flux toward methane precursors, whereas excessive CO2 could potentially limit Coenzyme M availability by overproducing Acetyl-CoA, highlighting the need for controlled dosing. Operational optimization identified a food waste-to-sludge ratio of 3:1 and an organic load of 5 g VS L-1 d-1 as optimal, yielding 0.159 L CH4/g VS, outperforming other tested ratios. These results demonstrate that CO2 injection can effectively improve substrate conversion efficiency and methane production while maintaining microbial stability. The findings provide practical guidance for high-load anaerobic digestion management and illustrate the potential of CO2 modulation to enhance system performance.