Amended Ferrozine Assay for Quantifying Magnetosome Iron Content in Magnetotactic Bacteria.

<i>Magnetospirillum gryphiswaldense</i> MSR-1 can biomineralize the magnetosome, nanoscale magnetite (Fe₃O₄) surrounded by a lipid bilayer, inside the cell. The magnetosome chain(s) enables MSR-1 to move along with the magnetic field (magnetoaerotaxis). Due to its unique characteristics, MSR-1 has attracted attention for biotechnological applications. During cultivation, not only the optical density but also the magnetosome content in MSR-1 should be monitored. The ferrozine assay had been utilized to quantify the iron content in magnetosomes. However, the effectiveness of the ferrozine assay on iron oxide nanoparticles is still unknown. Here, we examined the experimental factors, and the amended ferrozine assay demonstrates a recovery of 88.71% for Fe₂O₃ nanoparticles relative to the stock solution. Next, we apply the assay to analyze MSR-1 samples, which successfully reveals the difference in iron contents between magnetic and nonmagnetic MSR-1 samples and highlights the amount of MSR-1 cell density suitable for amended ferrozine assay. The assay further helps us examine the effects of centrifugation compared to magnetic separation (MS). The detection of residual magnetosomes in the supernatant indicates that MS remains a suitable method for collecting magnetosomes. We anticipate the amended ferrozine assay will facilitate research on MSR-1 by enabling investigators to measure iron content in cells in a fast, easy, and cost-effective manner.