Evaluation of Metal Accumulation in

Rare earth elements, comprising 17 elements including 15 lanthanides, are essential components in numerous high-tech applications. While physicochemical methods are commonly employed to remove toxic heavy metals (e.g., cadmium and mercury) from industrial wastewater, biological approaches offer increasingly attractive alternatives. Biomining, which utilizes microorganisms to extract valuable metals from ores and industrial wastes, and bioremediation, which leverages microorganisms to adsorb and transport metal ions into cells via active transport, provide eco-friendly solutions for resource recovery and environmental remediation. In this study, we investigated the potential of three recently identified lanthanide-binding proteins-SPL2, lanpepsy, and lanmodulin-for applications in these areas using single-cell inductively coupled plasma mass spectrometry (scICP-MS). Our results demonstrate that SPL2 exhibits superior characteristics for lanthanide and cadmium bioremediation. Heterologous expression of a cytosolic fragment of SPL2 in bacteria resulted in high expression levels and solubility. Single-cell ICP-MS analysis revealed that these recombinant bacteria accumulated lanthanum, cobalt, nickel, and cadmium, effectively sequestering lanthanum and cadmium from the culture media. Furthermore, SPL2 expression conferred enhanced bacterial tolerance to cadmium exposure. These findings establish SPL2 as a promising candidate for developing recombinant bacterial systems for heavy metal bioremediation and rare earth element biomining.