This work exploits a new microfluidic platform that is designed for optimal kinetic studies of electroactive biofilm metabolic activity. We used this system to investigate the activation energies and to determine reaction bottlenecks that were either related to acetate enzyme oxidation or electron transport for EABs of different ages (1 month and 4 months) under various flow rates (0.5 and 2 mL h -1 ) and acetate concentrations (10, 7 and 5 mM). Quantitative analysis of the results shows that the activation energy for electron transport in early-stage biofilms is significantly lower than that in mature biofilms. This indicates that in early-stage EABs, electron transport via cytochrome c enzymes is the primary bottleneck, whereas in mature biofilms, the bottleneck shifts from electron transport to acetate oxidation. This observation suggests that as biofilms mature, flow forces and the changes in internal structure result in changes to the mechanistic details of the metabolic process. Additionally, our work highlights the significant influence that flow rate and nutrient concentration have on the effective enzyme concentration, demonstrating that higher values of each enhance acetate permittivity into the EAB. Therefore, a strategy aimed at optimizing outputs from bioelectrochemical systems that include very mature EABs should focus on high acetate loading and shear flow to access otherwise inactive cells within the EAB interior.