Amended Ferrozine\nAssay for Quantifying Magnetosome\nIron Content in Magnetotactic Bacteria.

Magnetospirillum gryphiswaldense MSR-1 can biomineralize the magnetosome, nanoscale magnetite (Fe<sub>3</sub>O<sub>4</sub>) surrounded by a lipid bilayer, inside the cell.\nThe magnetosome chain(s) enables MSR-1 to move along with the magnetic\nfield (magnetoaerotaxis). Due to its unique characteristics, MSR-1\nhas attracted attention for biotechnological applications. During\ncultivation, not only the optical density but also the magnetosome\ncontent in MSR-1 should be monitored. The ferrozine assay had been\nutilized to quantify the iron content in magnetosomes. However, the\neffectiveness of the ferrozine assay on iron oxide nanoparticles is\nstill unknown. Here, we examined the experimental factors, and the\namended ferrozine assay demonstrates a recovery of 88.71% for Fe<sub>2</sub>O<sub>3</sub> nanoparticles relative to the stock solution.\nNext, we apply the assay to analyze MSR-1 samples, which successfully\nreveals the difference in iron contents between magnetic and nonmagnetic\nMSR-1 samples and highlights the amount of MSR-1 cell density suitable\nfor amended ferrozine assay. The assay further helps us examine the\neffects of centrifugation compared to magnetic separation (MS). The\ndetection of residual magnetosomes in the supernatant indicates that\nMS remains a suitable method for collecting magnetosomes. We anticipate\nthe amended ferrozine assay will facilitate research on MSR-1 by enabling\ninvestigators to measure iron content in cells in a fast, easy, and\ncost-effective manner.