The Fe(II) concentration was determined with ferrozine assay adapted from Stookey (45). Anaerobic cultures of S. oneidensis and E. coli strains were grown with Fe(III) citrate and fumarate, identical to the growth condition for the DEP-based screening as mentioned in the previous section. At each time point, one aliquot of each culture was centrifuged at 10,000 rpm for 5 min in the anaerobic chamber to pellet the cells, and 100 μl of the supernatant was acid-extracted in 900 μl of 0.5 M hydrochloric acid (HCl) to yield concentrations within the range of standard curves. The total iron concentration was determined by a separate acid extraction with 10% hydroxylamine hydrochloride in 0.5 M HCl for 24 hours. A hundred microliter of each acid-extracted sample was mixed with 900 μl of ferrozine reagent, which absorbs at 562 nm when chelating Fe(II). The ferrozine reagent contained 10 mM ferrozine (Sigma-Aldrich) in 500 mM HEPES (final pH = 7.0 adjusted by 2N NaOH). The absorbance of all samples was recorded at 562 nm with a UV Vis spectrophotometer (SHIMADZU, Japan) and was used to determine the formation of Fe(II) over time. The Fe(II) concentration in each culture was subtracted by abiotic iron reduction observed in medium-only controls at each time point. Standard curves were made from ferrous sulfate dissolved in 0.5 N HCl.

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