2.8. Determination of Siderophore Activity by Chrome Azurol-S Assay

Siderophore in the sample solution was determined using a modified chrome azurol-S (CAS) assay [22]. For the formulation of the CAS reagent, 0.75 mL of 2 mM CAS was mixed with 1 mL of 10 mM FeCl₃ (in 10 mM HCl) and 0.6 mL of 10 mM hexadecyltrimethylammonium (HDTMA). The final volume of the mixture was brought up to 10 mL with deionized water after the addition of 5 mL of 1 M MES (pH5.6) to make the CAS reagent. For the determination of siderophore, the peak fraction from the HPLC analysis (0.6 mL) was treated with 60 μL of 3% (w/v) 8-hydroxyquinoline (Sigma-Aldrich, St. Louis, MO, USA) in chloroform. The chloroform layer was removed after centrifugation (10,000× g, 5 min). The aqueous layer (0.5 mL) was mixed with the CAS reagent (0.5 mL) and 10 μL of 0.2 M sulfosalicylic acid. The reaction mixture was incubated for 1 h at 25 °C. The decrease in the absorbance at 650 nm (A₆₅₀) was monitored using a UV-vis spectrophotometer. The relative production of siderophore was calculated using the following equation.

where A^max stands for the A₆₅₀ value without siderophore and A^min for the A₆₅₀ value at the saturated siderophore concentration. A^sample represents the A₆₅₀ value in the presence of the siderophore sample.