Sampling and analytical procedures:

Wild type Azotobacter chroococcum strain B3 (ATCC strain 7486) and strain NCIMB 8003 (ATCC strain 4412) were grown aerobically in a modified Burk’s medium under diazotrophic conditions by shaking at room temperature as previously described.14,48 For HR-LC-MS analysis of siderophores, the availability of Fe was limited by addition of 100 M EDTA to 0.1 M FeCl3 for AC-B3, whereas a concentration of 5 M FeCl3 was added to achieve sufficient growth with AC-8003. To study the effect of Fe sources, Fe was added as (1) 100 M EDTA and 0.1 M FeCl3; (2) 100 M EDTA and 5 M FeCl3; and (3) precipitated amorphous Fe oxides. Bacterial growth was monitored at an optical density (OD) of 620 nm (OD620nm).

To follow concentrations of siderophores in AC-B3, 1 mL sample aliquots were taken at different times throughout the growth, filtered through 0.2 m syringe filters, acidified (0.1% acetic acid and 0.1% formic acid) and analyzed by direct injection on a single quadrupole LC-MS system (Agilent 6120). The analysis of all siderophores, except for crochelin A, was performed with a C18 column (Agilent Eclipse Plus C18 3.5 m, 4.6mm x 100 mm) and a gradient of solution A (water + 0.1% FA + 0.1% acetic acid) and B (acetonitrile + 0.1% FA + 0.1% acetic acid; gradient from 0–100% A over 30 min; flow rate of 0.8 mL/min). To achieve sufficient retention on the same C18 column, the analysis of crochelin A required the use of the ion-pairing reagent heptafluorobutyric acid (HFBA) added to solutions A (water + 0.05% HFBA) and B (acetonitrile + 0.05% HFBA) with a gradient from 0–100% A over 30 min; flow rate of 0.8 mL/min. The column outflow was diverted to waste for the first 5.25 min ensuring that the sample was completely desalted before introduction into the mass spectrometer. Siderophores were quantified by single ion monitoring (SIM) using a list of target m/z values of the identified siderophores. For quantification, LC-MS peak areas were determined and converted to concentrations by calibration with standards of vibrioferrin A,16 amphibactins ACA, S, and crochelin A. For catechol siderophores and azotobactins, LC-MS and UV-vis peak areas were determined using MassHunter software (Agilent). Relative peak areas were converted to concentrations by calibration with isolated standards of vibrioferrin, 2,3-dihydroxybenzoic acid (DHBA), azotochelin, protochelin, and azotobactin d. Seven technical replicates of a spent medium siderophore mix ‘‘standard’’ showed relative standard deviations of <10% when present at concentrations above 0.5 M. The detection limit of vibrioferrin A, amphibactins, and crochelin A in the supernatant sample was in the range of 0.02 to 0.10 M.