2.10 Chemical analyses
The concentrations of oxidized iron, nitrate, nitrite and ammonium ions were determined as previously described (Iino et al. , 2015a). After cultivation, culture fluids (100 µl) containing oxidized iron were acidified with 50 µl of 6 N HCl, and reduced with 100 µl of 1 M ascorbic acid for the quantification of total iron (ferrous and ferric ions). The iron ion concentration in each of the acidified solutions was determined colorimetrically using o -phenanthroline, as described by Sandell (1959). For the quantification of nitrate, nitrite, and ammonium ions, the culture fluids were centrifuged at 20,400×g for 10 min. The supernatant was recovered, and filtered through a 0.2-µm pore membrane filter. Nitrate, nitrite, and ammonium ions in the culture were quantified using a high-performance liquid chromatography (HPLC) system (model HIC-20Asuper; Shimadzu Corp.) equipped with a conductivity detector (model CDD-10ADsp), a Shim-Pack cation column (IC-C4), and a Shim-Pack anion column (IC-SA2). Corrosion products on the surface of iron coupons were also analyzed using an XRD analyzer with CuKa radiation ranging from 2θ = 5 to 100º at a scanning rate of 1º/min (Rint1500; Rigaku).