Microbial electrochemical cells (MxCs) are engineered biological systems that use microbial metabolism of anode-respiring bacteria (ARB) to catalyze electron transfer from a soluble electron donor to the anode via extracellular electron transfer (EET). Although several EET mechanisms (via direct contact, mediators, and conduction) have been proposed, understanding of EET in biofilm anodes generating high current density is limited. Recent findings suggested that electrical conduction would be a key EET pathway in MxCs producing high current density, in which biofilm conductivity (K_bio) would mainly regulate EET kinetics. However, there is no clear understanding of the influence of various environmental factors, such as anode potential, local pH, and substrate limitation in biofilm anodes on EET kinetics and K_bio. In addition, scalable, economical designs of MxCs producing high current density still need improvement for deployment of MxCs in field, such as multi-anode MxCs. Hence, the goals of this study were to systematically characterize the effects of (a) anode potential (E_anode), (b) local pH in biofilm anodes and (c) substrate limitations on EET kinetics and K_bio for a key fundamental aspect of MxCs, and develop scalable, economical MxCs using multi-anode configurations in an engineering aspect of MxCs.

A biofilm anode enriched with Geobacter spp. showed high K_bio (0.96-1.24 mS/cm) to E_anode change from -0.2 V to +0.2 V vs. standard hydrogen electrode (SHE), while the steady-state current density varied significantly in the MxC. Change of E_anode shifted population of Geobacter genus in the biofilm anode, influencing intracellular electron transfer (IET) kinetics. However, high K_bio was consistently kept in the biofilm at E_anode change. This result suggests that EET kinetics would be relatively insensitive to E_anode dynamics. A step-wise decrease in phosphate buffer concentration from 100 to 2.5 mM caused pH gradient of ~0.5 pH unit between the outmost and inmost layers of a biofilm anode, showing a pH of 6.5-6.7 near the anode in a thick (>100 m) biofilm. This pH gradient substantially dropped current density from 2.38 to 0.64 A/m² in an MxC, and K_bio decreased by 69% for the 2.5 mM phosphate buffer. These results imply that the metabolic activity of ARB inhibited by acidic pH is closely associated with conductive nature of biofilm anodes and EET kinetics. In a steady-state MxC, K_bio dynamically decreased from 0.53 mS/cm to 0.14 mS/cm during the long starvation (4-5 days) lacking exogenous electron donor. However, the poor K_bio was recovered to 0.55 mS/cm after acetate spiking, indicating that ARB’s activity profoundly influences K_bio and EET kinetics. A multi-anode MxC equipped with three anode modules showed a non-linear increase of current density to the number of anodes. The anode closest to a reference electrode (i.e., low ohmic energy loss) contributed to 65% of the overall current density of 9.15 A/m² from the multi-anode MxC, where Geobacter species were dominant at 87% and half saturation potential (-0.251 to -0.242 V vs. SHE) was lowest among all anode electrodes. In comparison, the current density from the other two anodes distant from the reference electrode was as small as 1.4-1.7 A/m², along with negligible population of Geobacter species. These results suggest that E_anode changed by ohmic energy losses in individual anodes can shift microbial communities, and lead to different electron transfer kinetics and current density on each anode.