3.2. Bio-Oxidation of Pyrite
The pyrite oxidation experiment was conducted for a period of 18 days. Total cell counts and DO concentrations during the experiment are shown in
Figure 4a,b, respectively. The cell density in reactor A remained relatively stable during the experiment, whereas the cell densities in reactors B and C increased during the first nine days (
Figure 4a). The cell density in reactor D decreased at the start of the experiment (from day 0 to 2), possibly due to the attachment of cells onto the ore. Thereafter, the cell density in reactor D increased gradually, and approached a similar level as in reactor C at the end of the experiment. It was unclear whether the initial cell attachment would have also occurred in reactors A, B, and C, in which the cell densities could have been too high to detect a measurable change as a result of attachment. The relative order of cell densities in the reactors remained A > B > C > D throughout the experiment. The DO concentration in all reactors was maintained at 7–8 mg L
−1 throughout the experiment (
Figure 4b). It is worth noting that severe foaming was observed in reactor A, which had the highest initial cell concentration of > 10
10 cells mL
−1. This foaming phenomenon may be caused by proteins released from broken cells due to the high shear forces inside the reactor (e.g., collision by the fine ores and the propeller) [
12]. As the cell concentration was very high, the likelihood of collision increased in reactor A compared to other reactors. The cells in reactor A may also have been starving due to the high cell to substrate ratio. This may have led to the death of some cells and release of organic compounds from the cells. Another possible explanation is the production of surface-active agents (biosurfactants) by the dense culture [
12].
The reactor pH values recorded via the Labview program and manually during the pyrite oxidation experiment are shown in
Figure 5a,b, respectively. The volumetric and gravimetric base consumptions are shown in
Figure 5c,d, respectively. The pH in reactors A, B, and C increased to between 1.46 and 1.50 within the first few hours after the start-up, likely due to the proton consumption during bio-oxidation of the ferrous iron in the growth medium (
Figure 5a,b). However, the initial rise in pH was only transient and the pH values in these reactors decreased to the set point of pH 1.4 within 10 h of operation (
Figure 5a). The decrease in pH was likely a combined effect of pyrite oxidation and generation of protons during ferric iron precipitation. Notable drifts (slight increases) in the readings of the pH probes were detected throughout the experiment. However, daily recalibrations of pH probes meant that the fluctuations did not impair the effectiveness of the feedback-control, which was unaffected and did not trigger any unintentional base addition. In fact, the base consumption measured by weight loss recorded for the experiment was in good agreement with the consumption recorded by the Labview program.
The base consumption in reactor D started immediately after the onset of experiment, as during this period the pH in D was lower than the set point (
Figure 5a,c). This may be due to the low cell concentration, and hence slow ferrous iron oxidation that did not consume as much acid as was generated in pyrite oxidation. After the initial pH correction, the pH in D gradually increased during the first 5 days, possibly due to ferrous iron oxidation. In all other reactors, the pH initially increased as a result of more rapid ferrous iron oxidation, and hence there was no NaOH demand at the beginning of the experiments in reactors A–C. NaOH demand in reactors A and B began at 13.3 h and 21.5 h (on day 0), respectively (
Figure 5c,d). A longer lag time (48 h) was observed for reactor C to start consuming NaOH (
Figure 5d), most likely due to the lower cell density contained in this reactor, and therefore lower ferrous iron oxidation rate.
The base consumption patterns recorded after the first day in the pyrite oxidation experiment could be generally divided into two distinct phases, phase 1: from days 1 to 8; and phase 2: from days 8 to 18. The base consumption during the first phase followed the order of reactor A > B > C > D. This trend was expected given that the cell densities in the reactors were in the same descending order. From days 8 to 18, the cell density in reactor D increased to a level that was similar to that of reactor C and by day 18 the cumulative NaOH consumption in reactor D was similar to that of reactor B. At the end of the experiment (day 18), the order of cumulative base consumption was reactor A > D > B > C and the rate of NaOH consumption in reactor D was the highest (
Figure 5c,d). The relationship between the amount of NaOH consumption measured by weight loss and the computer program in the pyrite oxidation experiment was linear as shown in
Figure 6.
Recorded NaOH consumptions were much lower than expected, only 2.71–3.47% of theoretical NaOH consumption, assuming complete pyrite oxidation (
Figure 7a). The actual NaOH consumption was also only 5.12–7.25% of that expected based on actual pyrite oxidation values derived from the mineralogical analysis of the residues from reactors A–D (
Figure 7a). According to the stoichiometry for complete pyrite oxidation (Reaction 7), 16 moles of OH
− (as NaOH) would be required to neutralise the protons generated for each mole of pyrite oxidised.
Considering the head ores used in the experiments contained 15% (
w/
w) pyrite, NaOH consumption of 80 g NaOH (per 100 g ore (dry wt.)) could be expected for complete pyrite oxidation. Hence, it was expected that the NaOH consumptions in all reactors (A to D) would be considerably higher. However, except for reactor D where the NaOH consumption appeared to be continuously increasing from day 8, all other reactors (A–C) showed signs of NaOH consumption slowing down at the end of the experiments (
Figure 5c,d).
The reason for the discrepancy between the recorded and theoretical NaOH consumption is unclear. However, weak linear correlations were obtained between pyrite (and sulfide) oxidation and the actual NaOH consumption (
Figure 7b). Hence, one may still consider NaOH consumption a rough surrogate indicator of pyrite oxidation for the specific head ore used in this study. Undoubtedly, more complicated reactions could have been involved in the process and as such, NaOH consumption should not be generalised for this purpose when different ores are tested. One factor could be the possible acid consumption of gangue minerals during the extended period of reactor operation as compared to the abiotic acid consumption test. Another factor contributing to the pH and NaOH consumption is the acid generated during precipitation of ferric iron as jarosite according to reaction 5, where A represents a monovalent cation such as ammonium (NH
4+), potassium (K
+), sodium (Na
+), or hydronium (H
3O
+) [
13].
Further understanding of the parameters or reactions that determine the rate and extent on the ferrous oxidation and ferric precipitation is essential, given the extent of ferric iron precipitation is dependent on the concentration of oxidised iron and the prevailing pH [
14]. Ferrous iron concentration, redox potential, and total soluble Fe and S concentrations during the experiment are shown in
Figure 8. The ferrous iron was nearly completely oxidised in reactors A–C within the first day, whereas in D near complete oxidation was reached on day 8 (
Figure 8a). Similarly, the redox potential in reactors A–C increased rapidly within a day and remained stable at approximately +650 mV thereafter until the end of the experiment. The increase in redox potential in reactor D was substantially slower. By day 10, the redox potential in reactor D was similar to those recorded for reactors A–C (
Figure 8b). The lower redox potentials in D during the first week of the experiment may also have caused a shift in the microbial community structure by allowing such microbial species that are sensitive to higher redox potentials to grow. For example,
A. ferrooxidans has been reported to have a higher growth rate at redox potentials below +470 mV (vs. Ag/AgCl), whereas
Leptospirillum spp. have a greater affinity for Fe
2+ and are less sensitive to inhibition by Fe
3+ at a higher redox potential [
15].
A. ferrooxidans can oxidise both Fe
2+ and reduced sulfur species whereas
Leptospirillum can only oxidise Fe
2+ [
9]. The possible shift in the microbial community structure may have increased the pyrite oxidation rates. However, since the microbial communities were not analysed at the end of the experiment, the possible shift in the community structure remains unconfirmed. It is also possible that indigenous microorganisms present in the non-sterilised ore may have become enriched towards the end of the experiment and contributed to the bio-oxidation.
As expected, the total soluble Fe and S concentrations in all reactors increased throughout the experiment (
Figure 8c,d). Initially, the total soluble Fe and S concentrations in reactors A and B increased at a similar rate. However, after the first week both total soluble Fe and S concentrations were highest in reactor B followed by reactors A, C, and D. By the end of the experiment the soluble iron and sulfur concentration in reactor D were similar to those in reactor B. However, total soluble Fe and S concentrations in reactors A and C were similar to each other, but lower than those in reactors B and D (
Figure 8c,d). Overall, the total soluble Fe and S results suggested that the kinetics of bio-catalysed pyrite oxidation was not proportional to the initial cell concentrations tested in this study (2.3 × 10
10–2.3 × 10
7 cells mL
–1). The higher cell density in reactor A may have enhanced jarosite precipitation, which in turn may have decreased the total soluble Fe and S concentrations (
Figure 8c,d). Extracellular polymeric substances (EPS) have been suggested to contribute to changes in solution chemistry that facilitates precipitation [
16].
DOC concentrations in the leach liquors at the end of the pyrite oxidation experiment were 76, 40, 27, and 30 mg L
−1 for reactors A, B, C, and D, respectively. The DOC concentration was highest in reactor A, followed by reactors B, D, and C. A photo of the supernatants of the reactor slurries collected at the end of the pyrite oxidation experiments is shown in
Figure 9. The supernatants collected from A (with the highest initial cell density) after mineral solids separation with short centrifugation at 3350×
g for 5 min appeared to be most turbid, probably due to higher cell densities. High concentrations of dissolved organic compounds may inhibit the activity of some bioleaching microorganism and also slow down abiotic pyrite oxidation with Fe
3+ [
17,
18,
19].
Figure 10 shows dried residues from the pyrite oxidation experiment, and masses of the recovered residues are shown in
Table 4. The brown colour on the surface of the residues from reactor A was likely due to cell biomass recovered along with the mineral residues (
Figure 10).
The elemental compositions of the residues from the pyrite oxidation experiment are shown in
Table 5. The Fe, total-S, and sulfide-S contents were lower in the residues than in the feed ore, with the lowest Fe and total-S contents detected for reactor D residue, followed by reactors B, A, and C, respectively. The sulfide-S was lowest for reactor B residue, followed by reactors D, A, and C, respectively. Total-C content was highest for reactor A residue, followed by B, D, and C, respectively. This is consistent with the higher biomass amount in A residue as observed visually (
Figure 9 and
Figure 10).
The mineralogical composition data of the feed and leached residues by Rietveld based QXRD conducted by CSIRO is listed in
Table 6. A reasonably good agreement was found between the previous MLA results of the feed ore and the QXRD results obtained for feed ore when analysed in conjunction with the leach residues. The feed ore was dominantly K-feldspar type minerals, with moderate amounts of illite type and kaolinite type clays, a moderate amount of pyrite and alunite, and trace amounts of Ti mineral anatase, jarosite, barite, quartz, and gypsum.
Based on the QXRD analysis, pyrite contents in all leach residues were lower than in feed ore, with the pyrite content being lowest in the residue from reactor D, followed by reactors B, A, and C, respectively. The pyrite contents in the leach residues from reactors A–D correlated well with the sulfide-S content observed by chemical analysis (
Figure 11). Dissolution of other major and moderate phases including K-feldspar, kaolinite, alunite, and illite were not obvious from the QXRD results (
Table 6). However, jarosite content in reactor A–D residues was increased as compared to the feed ore, which indicated jarosite formation under the reactor conditions. The jarosite content in the residue increased with increasing initial cell concentration. As mentioned previously the presence of higher number of cells and thus more abundant EPS may have enhanced jarosite precipitation [
16]. The presence of cations such as Fe
3+, Al
3+, K
+, Na
+, and NH
4+ in sulfate-based solution in slightly acidic conditions will encourage jarosite formation [
20]. Other than the soluble chemicals already present in the reactors (e.g., those present in the 0K medium and the dosed NaOH over the course of the experiment), additional cations may become available from the dissolution of pyrite, small amounts of K-feldspar minerals, and illite type minerals associated with the ore (
Table 6).
Results from the extraction of gold from residues of the pyrite oxidation experiment using 3.75 kg NaCN t
−1 varied from 71.91% to 87.78%, as shown in
Table 7. Gold extraction was highest for reactor D followed by reactors A, B, and C. By the end of the experiment, the effect of initial cell concentration was no longer evident and relatively high gold leaching was obtained for all residues as compared to ore that had not been bio-oxidised. For comparison, on average, cyanidation tests on the ore without bio-oxidation resulted in only 30–35% gold extraction (data not shown). Final cyanide concentrations after 24 h cyanidation were 0.055%, 0.055%, 0.075%, and 0.050%, and the corresponding cyanide consumptions were 2.93, 2.93, 2.63, and 3.00 kg NaCN t
−1 for residues from reactors A, B, C, and D, respectively.
A summary of the extent of pyrite oxidation in reactors A–D as estimated based on various parameters after one week and at the end of the experiment is shown in
Table 8. After the first week of the pyrite oxidation experiment, the NaOH consumption implied that the increasing cell numbers could enhance pyrite oxidation. Apart from reactor A, which had lower soluble Fe and S concentrations than B, the soluble Fe and S concentrations also increased with increasing cell numbers. However, at the end of the experiment (day 18), the effect of initial cell concentrations was no longer clear. Although reactor A still showed the highest NaOH consumption at the end of the experiment, the second highest consumption was recorded for reactor D, which had the lowest initial cell numbers. Reactor D also showed the second highest soluble Fe and S concentrations just after reactor B. Sulfide-S content of the residues indicated that reactor B would have the highest pyrite oxidation, whereas based on pyrite content and gold extraction the oxidation was more extensive in reactor D. At the end of the experiment, reactor C showed the lowest pyrite oxidation based on most of the indicators listed in
Table 8.
The fact that the highest initial cell concentration in reactor A did not result in the highest pyrite oxidation over the 18 day period could be due to the inhibitory effect of the elevated level of dissolved organic matter on microbial activity and pyrite oxidation [
17,
19]. Yacob et al. [
19] found that the rate of pyrite oxidation by ferric iron in sterile suspensions at pH 1.8 was reduced by 87% in the presence of dissolved organic compounds produced from the microbial cells. They also affirmed that such inhibition was attributed to the complexation reaction between the organic compounds and Fe
3+. Moreover, Marchand and Silverstein [
17] found that the rate and extent of pyrite oxidation by
A. ferrooxidans were limited by the presence of organic compounds, and that the growth of this iron oxidiser was notably inhibited by dissolved organic compounds.
At the start of the experiment, most cells in reactor A were likely active, which explains the rapid start-up and NaOH consumption. However, after a while the cells in reactor A may have been starving due to limited availability of substrates (ferrous iron and reduced sulfur compounds) released from pyrite. This may have caused some of the cells to die and release DOC. The foaming that was observed after a few days in reactor A may have been related to cell lysis as the DOC concentration in reactor A was higher than in other reactors at the end of the experiment.
Previous studies on the effect of initial cell concentration on the efficiency of bio-oxidation processes are somewhat limited in the biomining context. Okereke and Stevens [
21] and Boxall et al. [
7] evaluated the effect of cell concentration on ferrous iron oxidation rates at cell concentration ranges of 3 × 10
8–9 × 10
8 cells mL
−1 and 6.8 × 10
7–7.1 × 10
9 cells mL
−1 and temperatures of 10–30 °C and 30 °C, respectively, and reported increasing ferrous iron oxidation with increasing initial cell concentrations. The study by Okere and Stevens [
21] used a culture of
A. ferrooxidans and the study by Boxall et al. [
7] used a similar mixed culture composed of
A. caldus,
Ferroplasma acidarmanus/
acidiphilum,
L. ferriphilum, and
Sulfobacillus thermosulfidooxidans as used in the present study. Tambwe et al. [
22] evaluated the desulfurisation of high sulfur coal discards using inoculated an uninoculated columns. The inoculation was conducted with a mixed culture of
A. caldus and
L. ferriphilum at an initial cell concentration of 10
12 cells per ton of coal discards. Redox potential increased more rapidly in the inoculated columns than in the uninoculated control column that relied on native microbes. However, after approximately 50 days the redox potential in the uninoculated column reached similar levels to the inoculated columns. Sulfur removal as a result of bio-oxidation after 380 days was 61.6–63.5% in the inoculated columns and 60.1% in the uninoculated column [
22]. Although the applied system was different, namely an unsaturated column instead of a stirred tank reactor, the results are in agreement with those of the present study in that the prolonged operation of a sulfide ore bio-oxidation system somewhat masked the effect of the initial cell concentration on sulfide oxidation. Nevertheless, the findings of the present study revealed that increasing cell concentration is a viable strategy to enhance pyrite oxidation rate in reactors that are operated with a retention time of below 8 days.