Metabolic Flux-Based Regulation of Flexible Nodes Enhances Methane Yield in Anaerobic Digestion at Optimal Temperature

• • At 40 °C, up-regulating the acetyl-CoA flexible node increased methane flux by 48.5%, surpassing the 36.6% increase achieved by up-regulating the acetate node, indicating acetyl-CoA as the key regulatory point for maximizing AD methane production. • • The hydrogenotrophic methanogenesis pathway (R26) exhibited a 65.3% increase in flux upon acetyl-CoA up-regulation, while the acetoclastic pathway (R29) increased by 36.6%, highlighting differential pathway responses to metabolic node manipulation. • • The optimal temperature for AD methane production was determined to be 40 °C via FBA, providing a quantitative basis for process optimization. • • The FBA method requires only system input and output measurements to accurately compute intermediate fluxes, offering a cost-effective and reliable alternative to experimental metabolite tracking, with broad applicability to other fermentation processes.