Electrode plate-culture methods for colony isolation of exoelectrogens from anode microbiomes

Exoelectrogens play central roles in microbial fuel cells and other bioelectrochemical systems (BESs), yet their physiological diversity remains largely elusive due to the lack of efficient methods for the isolation from naturally occurring microbiomes. The present study developed an electrode plate-culture (EPC) method that facilitates selective colony formation by exoelectrogens and used it for isolating them from an exoelectrogenic microbiome enriched from paddy-field soil. In an EPC device, the surface of solidified agarose medium was spread with a suspension of a microbiome and covered with a transparent fluorine doped tin oxide (FTO) electrode (poised at 0 V vs. the standard hydrogen electrode) that served as the sole electron acceptor. The medium contained acetate as the major growth substrate and Coomassie Brilliant Blue as a dye for visualizing colonies under FTO. It was shown that colonies successfully appeared under FTO in association with current generation. Analyses of 16S rRNA gene sequences of colonies indicated that they were affiliated with genera Citrobacter, Geobacter and others. Among them, Citrobacter and Geobacter isolates were found to be exoelectrogenic in pure-culture BESs. These results demonstrate the utility of the EPC method for colony isolation of exoelectrogens.