3.3 Fate of nitrate during Fe0 corrosion
As has been observed in our previous study (Iino et al. , 2015a),P. denitrificans MIC1-1T grown in corrosion-test medium containing 10 mM nitrate corroded Fe0 to extents more than 20-fold higher than that in the aseptic control. When yeast extract in corrosion-test medium was substituted by D-glucose, Fe0 oxidation by P. denitrificans MIC1-1T was reduced by 50% probably due to the inhibitory effect of D-glucose on Fe0corrosion (Isa et al. , 2011). The three newly isolated nitrate-reducing strains, P. denitrificans AT004, P. denitrificans KGS048, and Prolixibacter sp. SD074, also oxidized Fe0 extensively (Table 3). Conversely, the two nitrate-non-reducing strains, Prolixibacter sp. NT017 andP. bellariivorans JCM 13498T did not show such an activity confirming the results from the electron-microscopic and electrochemical studies. None of the six strains enhanced Fe0 corrosion in the presence of sulfate in place of nitrate (Table 3).
In Fe0-foil-containing corrosion-test media inoculated with the nitrate-reducing strains, nitrate was reduced by 50% over 30 days (Table 3) similarly to the results in Table 2. On the other hand, the nitrite concentrations were lower, while the ammonium concentrations were higher in the Fe0-containing cultures (Table 3) compared to Fe0-free cultures (Table 2). From these results, and from the previous finding that nitrite chemically corrodes Fe0 (Alowitz and Scherer, 2002), we hypothesized that Fe0 was chemically oxidized to Fe2+and/or Fe3+ concomitantly with the reduction of nitrite to ammonium.
To clarify this point, time-course changes in the concentrations of nitrate, nitrite, and ammonium in the cultures of the four nitrate-reducing strains in the presence or absence of Fe0 were examined for four weeks. As shown in Fig. 6, a similar trend was observed among the four strains for the concentration changes of nitrate, nitrite and ammonium. In all the cultures (Fig. 6A–D, and F–I), the nitrate concentrations decreased sharply during the first week, followed by gradual decreases. Unexpectedly, the nitrite concentrations only increased slightly during the first week compared to the sharp decrease in the nitrate concentrations. Thus, the vast majority of nitrate reduced during the first 7 days was assimilated into biomass. This conclusion is in agreement with the results in Fig. 1; namely, the growth of the nitrate-reducing strains reached a plateau at day 7.
In the Fe0-non-amended cultures (Fig. 6A–D), the ammonium concentrations decreased during the first 2 to 4 days, indicating that a portion of ammonium present in corrosion-test medium was used as a nitrogen source during the growth of these strains. Subsequently, the ammonium concentrations increased. The nitrite concentrations in the same cultures increased continuously until the end of the cultivation period.
In the Fe0-amended cultures (Fig. 6F–I), the changes in the nitrite and ammonium concentrations were different compared with those in the Fe0-non-amended cultures. Compared to the ammonium concentrations in the Fe0-non-amended cultures, those in the Fe0-amended cultures were similar for the first two weeks, but higher in the last two weeks. On the other hand, the nitrite concentrations in the Fe0-amended cultures were highest on day 7, after which they declined. This decline was interpreted to be due to the chemical reduction of nitrite to ammonium coupled with the chemical oxidation of Fe0. In such a reaction, the reduction of one mole of nitrite to ammonium is coupled to the oxidation of either 2 mol of Fe0 to Fe3+, or 3 mol of Fe0 to Fe2+. To determine whether this stoichiometry was established, the relationship between the formation of oxidized iron and the consumption of nitrite was examined. As shown in Fig. 7, the increment in the concentrations of oxidized iron between day 7 and day X (Δ[Feb +]Day7_to_X) was calculated according using the following equation:
Δ[Feb +]Day7_to_X = [Feb +]DayX – [Feb +]Day7
where [Feb +]DayX is the sum of the concentrations of Fe2+ and Fe3+ on day X (X = 10, 14, 18, 21, 24 and 28). Because the changes in the nitrite concentrations between day 7 and day X can be expressed as:
[NO2]DayX – [NO2]Day7 = syn[NO2]Day7_to_X– deg[NO2]Day7_to_X
where syn[NO2]Day7_to_Xand deg[NO2]Day7_to_Xare the nitrite concentrations synthesized and degraded between day 7 and day X, respectively. Since syn[NO2]Day7_to_Xcan be estimated from the decrease in the nitrate concentration during this period:
syn[NO2]Day7_to_X= [NO3]Day7 – [NO3]Dayx
the nitrite consumption between day 7 and day X can be expressed as:
deg[NO2]Day7_to_X= [NO3]Day7 – [NO3]DayX + [NO2]Day 7 – [NO2]DayX
In Fig. 7, the bold lines show the fitting of the function a × deg[NO2]Day7_to_Xto the Δ[Feb +]Day7_to_Xdata points, where a is a fitting parameter. The best fittings were obtained with a = 1.25, 2, 2.3, and 1.85 for Fig. 7A, B, C, and D, respectively. Thus, the a values were often smaller than the expected values (between 2 and 3), probably because (i) the extraction efficiency of oxidized iron species from Fe0 foils may not be 100%, and/or (ii) nitrite was reduced not only chemically by Fe0 and Fe2+ but also biologically to either ammonium or nitric oxide. In any case, the results in Fig. 7 indicate that sufficient amounts of nitrite were consumed to account for the observed amounts of oxidized Fe0.
The chemical corrosion of Fe0 by nitrite under the current experimental setup was also examined. As shown in Table S4, nitrite oxidized Fe0 in a concentration-dependent manner.