Evidence of streamlined extracellular electron transfer pathway from biofilm structure, metabolic stratification, and long-range electron transfer parameters.

A strain of Geobacter sulfurreducens , an organism capable of respiring solid extracellular substrates, lacking four out of five outer membrane cytochrome complexes (strain extABCD + ) grows faster and produces greater current density compared to wild type grown under identical conditions. To understand cellular and biofilm modifications altered in extABCD + responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild type and extABCD + biofilms spanning 5 μm gaps. Our results reveal that extABCD + biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to wild type (based on increased rates of 15 N incorporation). We also observed an increased rate of electron transfer through extABCD + vs. wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multi-heme cytochromes streamlines electron transfer to electrodes. The combination of imaging, physiological and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than wild type. Importance Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4x higher current compared to wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.