3.2.1. Biomineralization (Sulfide) Characterization in Oxic and Anoxic Environments Using Scanning Electron Microscopy (SEM)
A modal investigation of the iron and sulfur mineral phases for the abiotic and biotic conditions was performed using SEM particle analyses. The proportion of sulfur and iron-bearing phases was determined from a total population of 1000 grains in which elemental ratios were defined. Particles with a high percentage of sulfur and iron were categorized as pyrite or potentially greigite, a precursor to pyrite framboids [40
], and compared to the proportion of iron oxide particles. Based on the elemental analyses for each particle, a strong contrast between the proportion of iron and sulfur-rich (<10% total modal percentage) particles were observed in the biotic incubations compared to the abiotic under anoxic conditions (Figure 1
). The proportion of sulfide particles observed in the biotic incubations (higher percentages of iron and sulfur) make up 4% of all the particles analyzed for the anoxic biotic incubations. SEM micrographs in Figure 1
show an aggregation of submicron microcrystals with a Fe:S ratio of 1:1. These measurements suggest the formation of monosulfides (e.g., Greigite, Mackinawite), which are precursors to pyrite formation and are known to be associated with microbial activity [40
]. These will most likely be replaced by pyrite if conditions persist and are formed from H2
S, which is produced by sulfsulfate reducing bacteria. [40
]. The chemical profiles suggest that H2
S is most likely being sequestered into aggregates of sulfide within the sediment. These particles in the abiotic incubation make up only 0.3% of the particles analyzed for the abiotic incubations and 1.8% from the initial top sediment sludge. This suggested that the microbes were vital in the formation of iron sulfides in storage environments. In the oxic condition, there were fewer particles with high sulfur concentrations for both biotic and abiotic incubations, though a large proportion had high iron concentration. These high iron, but low sulfur particles are most likely iron oxides.
3.2.2. Solid Phase Characterization and Metal Behavior, Using Principal Component Analyses (PCA)
Five solid phase extractions (e.g., water soluble, amorphous oxyhydroxides, weakly bound iron oxide phases, those prone to biological complexations, and strong acid extractable) were used to target metal solubility within the sludge material as a function of treatment. The PCA along with ANOVA analyses determined which metals within each mineralogical phase were susceptible to either biotic or abiotic geochemical alteration in both the presence and absence of oxygen. Only the amorphous oxyhydroxide phase were susceptible to microbial effects, while the other four phases were more strongly affected by the presence or absence of oxygen, based on differences between oxic and anoxic incubations. Those four geochemical phases were not altered by the presence or absence of microbial activity e.g., no new secondary mineral phases produced.
The targeted amorphous oxyhydroxide phase (i.e., easily reducible) within the sludge was susceptible to both microbial and atmospheric effects based on a PCA analysis and ANOVA. Analyses of all four mesocosm conditions showed distinct variation as each reported to separate quadrants (Figure 2
). 54% of the variance was explained by PC 1 and 27% of the variance was explained by PC 2, both of which are significant based on a 999-repetition row-wise bootstrap analysis. Both components had strong loadings from Fe, S, Al, and Ca with PC 2 suggesting an inverse relationship between Fe and S. Based on the scatter plot, it appears that PC2 shows microbial influence on the amorphous geochemical phase with the abiotic incubations both plotting positively, and the active incubations plotting negatively. The ANOVA analysis supports this as well, showing a significant variance of Fe concentrations in this phase which suggests microbial and atmospheric effects. The Fe concentrations in this amorphous phase in the biotic oxic incubation (1400 ± 300 mg/kg) are approximately 1.5× greater than within the abiotic. These phases include amorphous iron oxyhydroxides such as ferrihydrite, which is commonly found in AMD environments and is associated with microbes [43
]. Ferrihydrite can be formed directly by the oxidizing of Fe(II) by bacteria, or the bacteria can act as a nucleation site with mineralization of iron oxides encompassing both dead and living cells [43
]. Since amorphous iron oxyhydroxides are also involved with the adsorption of trace metals [44
], an unstable environment for these mineral phases would cause other types of contaminants common in the mine to be released in addition to Fe.
Other targeted phases included organically bound, water soluble, metals weakly bound to oxides, and strong acid extractable (total metals) phases. All followed a similar trend, with the atmospheric effects having a greater influence than the microbial effects. The variation in these geochemical phases was based on both PCA and ANOVA. For these phases, PC 1 explains >90% of the variance, and apart from the water-soluble phase, they all had strong loadings of Fe. In the water-soluble phase, PC 1 had loadings of Ca, S, and Mn. Concentrations within water-soluble fractions were lower in the anoxic mesocosm compared to the oxic for S (150 ± 15 compared to 250 ± 15 mg/kg) and Mn (10 ± 1 kg compared to 40 ± 5 mg/kg). This suggested higher stability in anoxic conditions concerning possible contaminants such as Mn and S. Other contaminants such as Zn (~0.2 mg/kg) and Mg (~25 mg/kg) were not significantly different between atmospheric effects, and Fe is not contained within any water-soluble phases. Compared to Mn and S, Zn, Mg, and Fe had no impact on the stability of the sediment based on the water-soluble phases.
The organically bound, weakly bound to oxides, and strong acid extractable phases all had strong loadings of Fe. The organically bound Fe concentration in the oxic mesocosm was 90 ± 20 (abiotic) and 210 ± 70 mg/kg (biotic) with no significant difference between abiotic and biotic in the anoxic conditions (2300 ± 300 mg/kg). Under anoxic conditions, Fe(II) will form colloids with organic matter and may be mobile in organic-rich systems based on a laboratory study [47
]. In a typical AMD or mine environment, organic matter may not be high, but previous studies suggest that mixing with soils under anoxic conditions may increase the chance of stable colloid formations, which may be easily transported downstream [40
]. Iron was not contained in the weakly bound oxides phase in the anoxic mesocosm, though the oxic had 2700 ± 200 mg/kg in the biotic and slightly less (2200 ± 300 mg/kg) for the abiotic. There were fewer oxides in the anoxic mesocosm overall, and iron may be preferentially adsorb to organic matter in the anoxic condition, rather than any oxides present.
Although Zn did not have strong loadings in any of the geochemical phases tested based on the PCAs, it is identified as an element of concern for the mine. Based on this study, up to 33 ± 5 mg/kg of zinc could be in the initial sediment across all mineral phases, though this decreases to 21–27 mg/kg after all incubations, suggesting that some Zn was possibly unaccounted for in the extractions or went under dissolution into the water column. Besides total extractable metals, the highest concentration of Zn was found in the extraction targeting sorbed metals to poorly crystalline phases, which was found to vary significantly by atmospheric condition (17 ± 4 mg/kg in the oxic and between 4–7 mg/kg in the anoxic). Zn will most likely be adsorbed or coprecipitated with oxyhydroxides [45
] in the oxic mesocosm. In this case Zn will be insoluble in water, based on the soluble phase phases’ extractions (<0.5 mg/kg measured). Other mineral phases had concentrations of zinc less than 2 mg/kg with no significant microbial or atmospheric effects.