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.