Fe(III) Mineral Encrustation Uncouples Respiration from Reproduction of the Nitrate-Reducing Fe(II)-Oxidizing Bacterium Acidovorax sp. BoFeN1

Fe(II) can be oxidized by mixotrophic nitrate-reducing Fe(II)-oxidizing (NRFeOx) bacteria to Fe(III), leading to mineral precipitation. However, the effects of this process on the metabolism and reproductive ability of Fe(II)-oxidizing bacterial cells have not yet been quantified. In this study, we investigated the effects of Fe(II) on the mixotrophic NRFeOx bacteria Acidovorax sp. BoFeN1 by performing cell plate counts, chemical analyses, scanning electron microscopy (SEM) imaging, live/dead fluorescence staining, and proteomic analysis. The results showed that nitrate-reducing Fe(II)-oxidizing bacteria Acidovorax sp. BoFeN1 undergo a striking physiological state in which respiration persists while cell reproduction collapses after their oxidation of Fe(II). Across 0.1-10 mM Fe(II), colony-forming units dropped by up to 10³-fold, yet acetate consumption remained largely unaffected, and nitrate reduction was even slightly promoted. SEM-EDS revealed extensive Fe(III) encrustation on cells, while live/dead staining showed intact inner membranes where nitrate reduction occurs. Adding an Fe(III) chelator, citrate, restored reproductive capacity without notable changes in Fe(II) oxidation or nitrate reduction, indicating that it was the products of Fe(II) oxidation, the secondary Fe(III) minerals, that impaired cell reproduction. The phenotypes observed in this study were supported by proteomic analyses, which showed that Fe(II) increased the abundance of proteins involved in nitrate reduction and the respiratory electron transport chain. These findings uncover a mineral encrustation-driven uncoupling between respiration and reproduction, suggest how natural organic ligands may buffer NRFeOx bacterial communities in Fe-rich anoxic environments, and point to cellular-level levers for mitigating denitrification failures and N₂O risks in engineered and natural systems.