Extraction and Detection of Structurally Diverse Siderophores in Soil

Although the biochemistry of bacterial and fungal siderophores has been intensively studied in laboratory cultures, their distribution and impacts on nutrient cycling and microbial communities in soils remain poorly understood. The detection of siderophores in soil is an analytical challenge because of the complexity of the soil matrix and their structural diversity. Liquid chromatography-mass spectrometry (LC-MS) is a suitable method for the sensitive analysis of siderophores in complex samples; however, siderophore extraction into liquid phases for analysis by LC-MS is problematic because of their adsorption to soil particles and organic matter. To determine extraction efficiencies of structurally diverse siderophores, spike-recovery experiments were set up with standards representing the three main siderophore classes: the hydroxamate desferrioxamine B (DFOB), the alpha-hydroxycarboxylate rhizoferrin, and the catecholate protochelin. Previously used solvent extractions with water or methanol recovered only a small fraction (< 35%) of siderophores, including < 5% for rhizoferrin and protochelin. We designed combinatorial chemical extractions (22 total solutions) to target siderophores associated with different soil components. A combination of calcium chloride and ascorbate achieved high and, for some soils, quantitative extraction of DFOB and rhizoferrin. Protochelin analysis was complicated by potential fast oxidation and interactions with colloidal soil components. Using the optimized extraction method, we detected alpha-hydroxycarboxylate type siderophores (viz. rhizoferrin, vibrioferrin, and aerobactin) in soil for the first time. Concentrations reached 461 pmol g(-1), exceeding previously reported concentrations of siderophores in soil and suggesting a yet unrecognized importance of alpha-hydroxycarboxylate siderophores for biological interactions and biogeochemical processes in soil.