Quantitative high-throughput approach to chalkophore screening in freshwaters

There is an increasing need to study the effects of trace metal micronutrients on microorganisms in natural waters. For Fe, small Fe-binding ligands called siderophores, which are secreted from cells and bind Fe with high affinity, have been demonstrated to modulate bioavailability of this critical nutrient. Relatively little is known about secretion of strong Cu-binding ligands (chalkophores) that may help organisms navigate the divide between Cu nutrition and toxicity. A barrier to environmental chalkophore research is a lack of literature on chalkophore analysis. Here we report the development of a quantitative, high-throughput approach to chalkophore screening based on a popular competitive-ligand binding assay for siderophores wherein ligands compete for metal in a chromogenic ternary complex of chrome azurol sulfonate-metal-surfactant. We developed the assay for high-throughput analysis using a microplate reader. The method performance is slightly better than that of comparable screening approaches for siderophores. We find that levels of other metals in natural samples may be capable of causing matrix interferences (a neglected source of analytical uncertainty in siderophore screening) and that for our method this can be overcome by standard additions. In this respect the high-throughput nature of the technique is a distinct advantage. To demonstrate practical use, we tested samples from field mesocosm studies that were set up with and without Cu and Fe amendments; we find trends in results that are logical in the environmental context of our application. This approach will be useful in areas such as risk assessment for a rapid survey of metal speciation and bioavailability; investigators who perform structural studies might also benefit from this approach to rapidly screen and select samples with high Fe/Cu binding capacity for further study.