Sulfur oxidation precedes ferrous iron oxidation with dispersed
sulfur
We serendipitously discovered an alternative sulfur formulation,
supplied by a commercial vendor as colloidal sulfur, which surprisingly
dispersed readily and evenly upon addition to the media to create a
homogenous mixture (Fig. S4). The new formulation, referred to as
d-sulfur, enabled A. ferrooxidans to exhibit starkly different
consumption patterns for iron and sulfur as compared to c-sulfur even
when added at a low concentration of 1.0 g/L. In cultures with d-sulfur,
the pH of the solution rapidly decreased to a minimum whereas little
ferrous iron oxidation occurred. The bulk of the iron oxidation and the
associated pH increase occurred after 36 h (Fig. 2A). When c-sulfur was
used instead at 1.0 g/L, we observed the commonly recognized behavior of
ferrous iron oxidation followed by sulfur oxidation, indicating that the
inoculated cells did not all adsorb to the c-sulfur surface (Fig. 2A).
These results suggested that the physical properties of d-sulfur were
special in allowing A. ferrooxidans to utilize the sulfur
quickly, indicating that the observed consumption order was influenced
by the hydrophobicity of the sulfur particles.
At higher concentrations of d-sulfur, the time needed for the completion
of the ferrous iron oxidation was increasingly prolonged, demonstrating
that additional sulfur repressed iron oxidation (Fig. 2B). At the two
highest concentrations tested of d-sulfur, 5.0 g/L and 10.0 g/L, ferrous
iron concentration initially decreased and then reversed to increasing
over the course of hours (Fig. 2B). The media pH continued to decrease
well below the critical pH of 1.3, which is suggested to inhibit iron
oxidation, reaching a final pH of 0.73 ± 0.01 and 0.63 ± 0.01 at 168 h
respectively, for d-sulfur concentrations of 5.0 and 10.0 g/L (Fig. 2B).
With the formulated d-sulfur, the reduction of ferric iron was observed
quickly than previously reported as the consumption of dispersed sulfur
led to the rapid acidification of the media under aerobic conditions.
The color and different physical properties of d-sulfur compared to
c-sulfur indicated that it had been chemically processed prior to being
sold to us by the vendor (Fig. S4). Based on its appearance and
properties, we hypothesized that the d-sulfur was a water dispersible
granule used in agricultural applications, produced by emulsifying
sulfur with a lignosulfonate salt (Eller & Person, 1969). As the exact
composition of the commercial d-sulfur was unknown, we studied several
classes of surfactant compounds and developed a simple sulfur
formulation to enable the preferential consumption of elemental sulfur
prior to ferrous iron oxidation in A. ferrooxidans. Numerous
dispersant candidates were tested, including calcium lignosulfonate,
other types of sulfonated compounds, a glycolipid, Tween-20, andA. ferrooxidans cell lysate. All of these compounds dispersed the
c-sulfur into water, and dry formulated powders were obtained (Fig. S4).
The calcium lignosulfonate dispersed sulfur, referred to as lig-sulfur,
dispersed into AFM1 medium similarly to d-sulfur, unlike c-sulfur which
immediately phase separated after vigorous vortexing with the medium
(Fig. S4).
Once these powders were redispersed into AFM1 medium, only a few of the
formulations tested displayed altered sulfur utilization behaviors.
Little to no growth was detected with sulfur dispersed with sodium
naphthalene sulfonate, poly-(sodium 4-styrenesulfonate), and Tween-20 as
no media clarification or color changes were observed when added to the
media, suggesting that these chemicals were incompatible with A.
ferrooxidans . The substrate utilization behavior at 1 g/L lig-sulfur in
conjunction with iron closely mimicked that of d-sulfur (Fig. 2C).
Furthermore, the growth of the cells on 10 g/L of lig-sulfur also
exhibited the transition from ferrous iron oxidation to ferric iron
reduction demonstrating that the formulation we developed induced a
similar response from A. ferrooxidans as d-sulfur (Fig. 2C). When
the glycolipid sucrose palmitate was used to disperse sulfur,
improvements to sulfur oxidation were achieved, but the effects were
less pronounced than with the lignosulfonate. Comparison of the 20 (wt
%) sucrose palmitate to the 33.3 wt % sucrose palmitate dispersed
sulfur cultures demonstrated that there is a critical point for
dispersion where the dispersed sulfur becomes bioavailable for oxidation
by the cells so that sulfur oxidation largely precedes ferrous iron
oxidation. The final pH for the 33.3 wt % sucrose palmitate sulfur was
lower at the end of ferrous iron oxidation as compared to the 20 wt %,
suggesting that there was faster utilization of sulfur when the
additional sucrose palmitate was incorporated into the formulation (Fig.
S5). Interestingly, we also found that the components of A.
ferrooxidans cell lysate were sufficient for dispersion and enabled
elemental sulfur oxidation before ferrous iron oxidation (Fig. S5).
We further characterized the aerobic ferric iron reduction that was
potentially detected with growth on high concentrations of dispersed
sulfur. To minimize the effects of ferrous iron oxidation by the cells,
the ferrous iron was replaced with ferric iron, so that the metabolic
energy for growth would have to come from the oxidation of d-sulfur. In
the abiotic controls, we found that the d-sulfur itself results in some
reduction of ferric iron, and is a primary contributor of the ferrous
iron measured initially (Fig. 3). In the cultures with A.
ferrooxidans , some additional ferrous iron than the control is measured
for the first 40 h as the cells oxidize the d-sulfur, but a major
portion of ferric iron reduction is observed as the cultures became more
acidic below the pH threshold of 1.3. Over a third of the initial ferric
iron in solution was reduced by 168 h when the pH dropped to the acid
tolerance limit of A. ferrooxidans (Fig. 3). These data emphasize
the observation that ferric reduction behavior continues until cell
growth is inhibited by the extremely low pH.