Metabolic Flux-Based Regulation of Flexible Nodes Enhances Methane Yield in Anaerobic Digestion at Optimal Temperature
Anaerobic digestion (AD) is an environmentally friendly biochemical technology for waste treatment and renewable energy production, yet its methane conversion efficiency remains suboptimal. This study employed flux balance analysis (FBA) to determine the optimal temperature for methane production in AD, and subsequently regulated key flexible nodes in the metabolic pathway to maximize methane flux. At the optimal temperature of 40 °C, up-regulating the acetyl-CoA flexible node increased methane flux by 48.5%, while up-regulating the acetate node increased it by 36.6%. The higher improvement via acetyl-CoA regulation is attributed to the fact that 40 °C is unfavorable for hydrogen-producing acetogenic bacteria, making acetyl-CoA the critical control point. These findings demonstrate that flexible node regulation can overcome the limitations of temperature optimization alone. The FBA methodology provides a reliable, cost-effective approach for optimizing target product yields in AD and other fermentation systems, requiring only input and output measurements to resolve intermediate metabolic fluxes.