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).