Trace metal–buffered Fraquil medium (Morel et al., 1975) containing 10 μg mL−1 spectinomycin was used in the experiments of various iron www.selleckchem.com/products/Cilomilast(SB-207499).html concentrations. Fraquil medium containing various levels of total iron (FeCl3) from 10 to 3000 nM was prepared. Iron exists mainly (92.15%) in the

form of Fe–EDTA complexes ([Fe–EDTA]− and [FeOH–EDTA]2−) in the medium. The free ferric ion concentration (pFe = −lg [Fe3+ free ferric]) and Fe(III)’ (the sum of the major inorganic iron species) were calculated using mineql+ software (Schecher & McAvoy, 1992): 10 nM (pFe 21.7, Fe(III)’ = 0.20 pM), 15 nM (pFe 21.5, Fe(III)’ = 0.30 pM), 30 nM (pFe 21.2, Fe(III)’ = 0.60 pM), 50 nM (pFe 21.0, Fe(III)’ = 1.01 pM), 70 nM (pFe 20.8, Fe(III)’ = 1.42 pM), 100 nM (pFe 20.7, Fe(III)’ = 2.04 pM), 300 nM (pFe 20.2, Fe(III)’ = 6.39 pM), 500 nM (pFe 20.0, Fe(III)’ = 11.12 pM),

ACP-196 order 1000 nM (pFe 19.6, Fe(III)’ = 25.05 pM), 3000 nM (pFe 18.8, Fe(III)’ = 151.45 pM). Early exponential phase cells grown for two generations in Fraquil medium with 100 nM Fe3+ were collected by centrifugation at 3900 g for 5–8 min, washed three times with iron-free Fraquil medium, inoculated into 300 mL polycarbonate flasks containing 100 mL Fraquil medium with various concentrations of Fe3+ (initial inoculum density OD730 nm = 0.06), cultured at 30 °C under continuous illumination of 25 μmol photons m−2 s−1 with shaking (135 r.p.m.), and sampled to measure luciferase activity, respectively, after 0, 12, 24, and 48 h. To minimize trace metal contamination, all culture materials were soaked in 10% HCl and rinsed thoroughly with Milli-Q water prior to use, and all solutions were prepared with Milli-Q water. To optimize the measurement parameters, the luciferase activity of bioreporter Palr0397-luxAB in Fraquil medium with 10, 100, and 1000 nM Fe3+ was measured at different inoculum cell densities (OD730 nm  = 0.02,

0.04, 0.06, 0.08, and 0.1), and different concentrations isometheptene of nitrogen (1, 10, 100, 300, and 900 μM), phosphorus (0.1, 1, 10, 30, and 90 μM), Co2+ (0.1, 0.5, 2.5, 7.5, 22.5, and 250 nM), Mn2+ (0.92, 4.6, 23, 69, 207, and 2300 nM), Zn2+ (0.16, 0.8, 4, 12, 36, and 400 nM) and Cu2+ (0.04, 0.2, 1, 10, 50, and 100 nM), respectively. The bioreporter cells were cultured in Fraquil medium as stated previously and sampled to measure luciferase activity after 12 h. Luciferase activity was measured according to Elhai & Wolk (1990). One microliter n-decanal (Sigma) was added into 1 mL bovine serum albumin (20 mg mL−1) and vortexed to obtain the reaction substrate. One milliliter of bioreporter was supplemented with 100 μL reaction substrate, gently mixed, and measured in a tube luminometer (Junior LB 9509) after dark treatment for 2 min to record relative light units (RLU). Luciferase activity was calculated as relative luminescence units per microgram of chlorophyll a.