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.