Single-Cell Mass Spectroscopic Analysis for Quantifying Active Metabolic Pathway Heterogeneity in a Bacterial...

Microbial electrochemical catalysis based on respiratory reactions coupled with extracellular electron transport (EET), which is critical for bioenergy applications, strongly depends on the biocompatibility of the electrode material. However, the comparison of materials for such physiological responses has been difficult because of the lack of a quantitative assay for characterizing cellular metabolism at the electrode surface. Here, we developed a single-cell analysis method specific for the cells attached to the electrode to quantify active metabolic pathway heterogeneity as an index of physiological cell/electrode interaction, which generally increases with metabolic robustness in the microbial population. Nanoscale secondary ion mass spectrometry followed by microbial current production with model EET-capable bacteria, Shewanella oneidensis MR-1 and its mutant strains lacking carbon assimilation pathways, showed that different active metabolic pathways resulted in nearly identical ¹³C/¹⁵N assimilation ratios for individual cells in the presence of isotopically labeled nutrients, demonstrating a correlation between the ¹³C/¹⁵N ratio and the active metabolic pathway. Compared to the nonelectrode conditions, the heterogeneity of the assimilated ¹³C/¹⁵N ratio was highly enhanced on the electrode surface, suggesting that the metabolic robustness of the microbial population increased through the electrochemical interaction with the electrode. The present methodology enables us to quantitatively compare and screen electrode materials that increase the robustness of microbial electrocatalysis.