Almost a decade has passed since the rediscovery of microbial fuel cell research, yet the power generation of these devices has not advanced. This is mainly due to the research being focused solely on the improvement of power generation rather than on a fundamental understanding of electron transfer processes and their limitations in the biofilms grown on the anode and cathode.  The chemical and electrochemical gradients in these biofilms play a critical role in electron transfer rates between cells and a solid electron acceptor or donor. Our research group has developed and used novel microelectrodes and in situ Nuclear Magnetic Resonance (NMR) imaging techniques to investigate electron transfer mechanisms in biofilms. The newly developed microelectrodes were successfully operated on polarized surfaces. Both NMR and microelectrode technologies allowed us to quantify metabolic and chemical variations in biofilms while the cells respired on polarized electrode surfaces. We combined our techniques with two new techniques – rotating disk electrode (RDE) and electrochemical quartz crystal balance (eQCM).  The addition of both these techniques allowed us to differentiate mass transfer limitations from intra-biofilm electron transfer processes. When we attempted to identify limitations in anodic biofilms, first, we addressed the importance of local acetate concentrations within G. sulfurreducens biofilms using a novel acetate microelectrode which has better resolution than NMR. The microelectrode work confirmed our previous NMR findings and generated profiles on the order of µM. We should note that the microelectrode method developed is significantly more economical to use than NMR and can facilitate rapid in situ measurements of acetate in anoxic biofilms.  Second, we integrated our previous RDE method with our eQCM methods to show that, on both a per current basis and a per mass basis, biofilm capacitance is a better indicator of biofilm performance than conductance. In particular, we compared the capacitance/conductance relationship of G. sulfurreducens biofilms to those of both polyaniline films and diffusing flavin systems. The results indicated that the tandem increase in capacitance and conductance is unique to the extracellular electron transfer mechanisms operating in G. sulfurreducens biofilms. Our local potential and current measurements indicated that the upper layers of the biofilms are always under a reducing condition. This knowledge also confirmed that the amount of cytochromes in the biofilm is not high enough or the biofilm conductivity is not sufficient to generate an oxidizing condition near the top of a biofilm. Finally, we extended the role of excess surface area in the scale-up of bioelectrochemical systems by examining biomass accumulation under this condition. We found that biomass continues to accumulate on the excess surface area electrodes with net arithmetic growth rates and acetate consumption during steady-state current. The linear growth rates constrained by ion transport limitations that we found may be important factors in understanding the current generation using high-surface-area 3D electrodes with advection.  In conclusion, we demonstrated the importance of the metabolic inactivity of layers within G. sulfurreducens biofilms using microelectrodes and NMR. In addition, we verified these findings using electrochemical impedance spectroscopy (EIS) by demonstrating the pseudocapacitive behavior of biofilm impedance. We also demonstrated the feasibility of directly measuring biofilm inefficiencies via intra-biofilm electron transfer rates and local current depth profiles.