In this section, we analyze the EA eff values that are plotted in Figure to understand more about the reaction bottleneck steps inside our optimized BES. In previous reports, certain EET steps were found to be the main reaction bottleneck at all growth stages. In this work, we attempted to look again at this issue, but with the expanded scope and precision provided by the developed 3-electrode microfluidic BES microfluidic bioanalytical system with a perpendicular flow orientation. The latter microfluidic advantage was used to specify the concentrations and homogenize the concentration gradients across the EAB outer surface, but it also gave us the unique opportunity to probe the effect of applied fluid forces and acetate concentrations. Perhaps, most importantly, the ability to culture the EAB for longer than 1 month enabled fundamental improvements to kinetics in highly matured systems to be evaluated for bottlenecks for the first time. We started our analysis at a flow rate of 2 mL h -1 on the 1-month EAB. The low EA eff measured under these conditions indicated that EET through cytochrome c enzymes was the reaction bottleneck step. It should be noted that nano-pili may still be directly conducting electrons, but that such conduction may not be the principle or the limiting EET process. Lowering the flow rate to 0.5 mL h -1 resulted in increased EA eff , likely because of an onset of diffusion limitations due to increasing boundary layers around the EAB that limit the enzyme-catalyzed oxidation of acetate. This effect was partially reversed by increasing the concentration to 10 mM, resulting in a shift back toward EET limitations, as indicated by the reduced EA eff . After 4 months, the system had an increased EA eff and demonstrated a different response to flow rate and concentration. The higher EA eff points to a shift to a bottleneck reaction step different than the EET limitations observed after 1 month. We attribute this to the older EAB having established a more robust EET network and a greater capacity to process high acetate concentrations. This ability for older G. sulfurreducens EABs to benefit from higher acetate concentrations was recently observed. It should be noted that, although the rate limitation had been shifted away from EET to a different mechanism, the EA eff is a weighted average of all individual EA values. Thus, while the older EAB had already become less limited by EET, increased flow rate and acetate concentration appeared to shift the reaction bottleneck even further away from EET.