Growth of A. ferrooxidans on untreated sulfur and ferrous
iron
We first investigated how the adsorption capacity of the c-sulfur
surface affected the population behavior and apparent substrate
utilization of A. ferrooxidans. These experiments were conducted
in the presence of both ferrous iron and elemental sulfur, which are the
two energy sources most commonly associated with this species. The
microbial oxidation of ferrous iron and sulfur by A. ferrooxidansimpacts the pH of the culture where ferrous iron oxidation is
accompanied by an increase in pH because of proton consumption through
oxygen reduction and the oxidation of sulfur results in a decrease in pH
because of sulfuric acid production.
Under a high initial cell density of an optical density at 600 nm
(OD600) of 0.05, corresponding to a cell density of 4.2
× 108 cells/mL, the oxidation of 72 mM of iron in the
medium without untreated colloidal sulfur, referred to as c-sulfur,
occurred rapidly, and the consumption of ferrous iron reached completion
by 18 h (Fig. 1A). When 10 g/L of c-sulfur was added under the same
conditions, the oxidation of ferrous iron reached completion by 24 h,
displaying a small but noticeable delay compared to when c-sulfur is not
present (Fig. 1A). Despite the oxidation of ferrous iron reaching
completion before c-sulfur, the change in pH associated with the
oxidation of the same concentration of iron differed when c-sulfur was
added. The average maximum pH for the three cultures without c-sulfur
was 2.20 ± 0.01, while the average maximum pH for the three cultures
with c-sulfur was 2.02 ± 0.01 (Fig. 1A). When c-sulfur was added, the
planktonic cell population in the cultures initially reduced as some of
the population adsorbed to the sulfur surface and was not detected.
However, the remaining planktonic cells rapidly multiplied in the first
24 h corresponding to the consumption of the readily available energy
obtained from soluble ferrous iron, which requires no complex processing
by the cells (Fig. S3). This observation led us to infer that the high
density of cells added exceeds the adsorption capacity of the c-sulfur
surface and that adsorbed cells did not contribute to iron oxidation as
the same total number of cells were added in both conditions.
Furthermore, the pH data suggested that A. ferrooxidans cells
pre-cultured with ferrous iron were capable of simultaneously oxidizing
small amounts of c-sulfur to produce sulfuric acid, leading to a lower
maximum pH in culture.
Thus, we studied the population behavior of these cells using an order
of magnitude lower initial cell density of OD600 of
0.005, corresponding to a cell density of 4.2 × 107cells/mL. We expected a significant proportion of the inoculated cells
to be adsorbed to the surface under these conditions and the apparent
lag phase would be extended as more cell divisions would be needed to
cover the available surface sites on c-sulfur before planktonic cells
became present in appreciable numbers to oxidize ferrous iron. While the
cells took longer to consume the ferrous iron without c-sulfur, reaching
completion by 34 h, we were surprised to find that the lag phase before
iron oxidation was significantly prolonged, taking 120 to 144 h to reach
completion (Fig. 1B). The average maximum pH for the three cultures
without c-sulfur was 2.20 ± 0.01, unchanged from that of the high
initial cell density condition (Fig. 1B). These data demonstrated that
the oxidation of iron dominates the pH change and was unaffected by the
number of cells catalyzing this reaction. The average maximum pH for the
three cultures containing c-sulfur was lower at 1.91 ± 0.02, and the
slight pH decrease detected before the onset of iron oxidation suggested
that some sulfur oxidation prior to iron oxidation occurred (Fig. 1B). A
small but non-zero concentration of planktonic cells were detected
during the initial incubation period on c-sulfur prior to iron
oxidation, which shows the active process of cell adsorption to the
particles (Fig. S3). The number of planktonic cells began to rapidly
increase at 120 h when ferrous iron oxidation was detected (Fig. S3).
An initial cell density of OD600 of 0.001, corresponding
to a cell density of 8.3 × 106 cells/mL, was used to
confirm that the lag phase depended primarily on cell loading. Under
these conditions, the onset of iron oxidation occurred around 192 h,
later than that was observed for the initial OD600 of
0.005 (Fig. 1C). The effect of modulating the ferrous iron concentration
on the lag phase in medium containing 10 g/L c-sulfur was also
evaluated. We found that changing the ferrous concentration from 72 mM
to 100 µM had small effect on the time taken to reach exponential phase.
However, elimination of all ferrous iron for metabolism and trace
nutrients prevented A. ferrooxidans from growing (Fig. 1C). The
slight acidification of the medium was approximately similar to that for
abiotic controls under the same condition corresponding to the slight
evaporation of water over time. The concentrations of ferrous iron had
pronounced effects on the late exponential phase of growth, particularly
as each of the conditions that passed the ferrous iron oxidation
threshold of pH 1.3. The rate of media acidification decreased earliest
for cultures with the lowest concentrations of iron, while the cultures
with 72 mM of iron more rapidly reached a lower pH (Fig. 1C). Ferric
iron reduction activity below pH 1.3 was also detected as the ferrous
iron concentration started to increase as sulfur oxidation continued
(Fig. 1C).
Visual changes in the cultures with a starting OD600 of
0.005 were documented at key time points (Fig. 1D). Initially, the
hydrophobic c-sulfur phase separates to the air-water boundary and
sticks to the glass walls. For the replicate which happened to have the
earliest onset of iron oxidation, a dispersed mixture was produced by 96
h prior to consumption of ferrous iron. At 120 h, when the ferrous iron
is depleted, the medium took on the dark orange color of ferric iron
which became paler over time as the pH of the solution decreased. These
visible markers were similarly noted for the other two replicates at
relevant times, unveiling that iron-adapted A. ferrooxidans cells
can process c-sulfur into a bioavailable and accessible form without
significant iron oxidation.