3.2. Solution Behavior During the Leaching of Lepidolite
Nonactivated lepidolite ore and samples with different mechanical activation times were leached individually in the eutrophic, oligotrophic bioleaching, and sterile control groups. In the initial stage of leaching (0–2 days), a slight increase in pH was observed in almost all the groups. (
Figure 6a), which can be attributed primarily to the exchange between Li
+/K
+ and other ions in lepidolite and H
+, as well as the acid dissolution reaction of lepidolite, which consumes a small number of protons. The pH increase trend was slowest in the eutrophic groups, and there was no increasing trend throughout the entire experiment in the nonactivated eutrophic groups. This might be explained by the preferential reaction of the microorganisms with pyrite, coupled with a higher rate of acid production than acid consumption by the lepidolite. In all the eutrophic bioleaching groups, the pH decreased, primarily due to the oxidation of pyrite by acidophilic microorganisms, which generated sulfuric acid and provided an abundant source of H
+ ions (Equations (2) and (3)). Notably, the eutrophic groups containing pyrite were subjected to nonuniform pH changes due to the presence of microbial ferrous oxidation-consuming protons (Equation (4)) and the formation of secondary minerals in the later stage. In contrast, in the oligotrophic bioleaching groups and sterile control groups, the pH of the leachate remained relatively stable at approximately 2.0 throughout the leaching period.
The change in the ORP in the leaching solution was affected by microbial metabolic behavior and reactants (
Figure 6b), especially the changes in the concentrations of Fe
2+ and Fe
3+. The eutrophic groups, before and after mechanical activation, initially had an ORP of approximately 420 mV, which continuously increased to approximately 700 mV within 0–8 days and then tended to stabilize. Acidophilic microorganisms can oxidize Fe
2+ to Fe
3+, resulting in an increase in the [Fe
3+]/[Fe
2+] ratio in the solution, which resulted in a rapid increase in the ORP. After 8 days, the concentrations of Fe
2+ and Fe
3+ reached a state of equilibrium, thereby maintaining a stable ORP. All oligotrophic groups and sterile control groups exhibited only minimal changes throughout the entire experiment.
During bioleaching in both the eutrophic and oligotrophic groups, there were notable differences in the changes in the free acidophilic microorganism concentration over time (
Figure 6c). In all the eutrophic bioleaching groups, there was no significant decrease in the microorganism concentration, indicating that the leaching groups are adaptable and that the oxidation of pyrite can promote the growth of microorganisms [
14]. In oligotrophic groups, the variation in microorganism concentration was relatively slower than that in eutrophic groups; however, overall, there was still an increasing trend. This occurred because there is no additional energy substrate in oligotrophic groups, whereas lepidolite contains small amounts of elements such as Fe and S to provide energy for microorganisms. Sedlakova-Kadukova et al. [
7] reported that microorganisms may be forced to use the nutrients needed for their life directly in the leaching solution and lepidolite. In addition, the studies of Rezza et al. [
4] also showed that metabolites such as gluconic acid, citric acid, and extracellular polymers in the leaching solution are related to bioleaching, and the important factor in enhancing this process may be the adaptation of microorganisms to oligotrophic environments. Notably, the concentration of microorganisms in the solution appeared to be positively correlated with the duration of mechanical activation. After 150 min of activation, the concentration in the oligotrophic leaching groups reached approximately 35 times the initial inoculation volume. This could be attributed to the generation of fresh surfaces from mechanical activation, which provides more contact sites between microorganisms and lepidolite, thereby enhancing the energy availability for microbial growth and metabolism.
In lepidolite, it is predicted that Li is bound between layers of AlO
6 in an octahedral structure and SiO
4 in a tetrahedral structure, acting predominantly as a charge supplement to ions. Therefore, the dominant bonding force is predicted to be highly ionic in these crystal structures [
19]. Lithium, aluminum, and silicon ions are crucial in the crystal structure of lepidolite. Therefore, the contents of lithium, aluminum, and silicon were detected mainly during the lepidolite leaching process. The trend of [Li
+] in the solution in the leaching process (
Figure 6d) indicated that [Li
+] was proportional to the duration of mechanical activation. This is because mechanical activation can cause cracks in the ore particles to increase the access of the microbial cells and electron shuttles to the minerals, thereby promoting the leaching of ions from the lepidolite. Moreover, under identical activation durations, the eutrophic groups presented the highest concentration of [Li
+], followed by the oligotrophic bioleaching groups, whereas the sterile groups presented the least effective lithium leaching capability. The variation trend of [Al
3+] (
Figure 6e) in the solution was consistent with that of [Li
+]. The concentration changes of silicon were more complex (
Figure 6f), which might be attributed to the formation of silicate minerals due to the combination of most metal ions with free silicate ions [
16], resulting in a change in the solubility of silicon, as shown in Equation (5), where M stands for metal ions. The silicate minerals subsequently decomposed in the acid-leaching group, and some silicate components were released into the solution.
Furthermore, the variations in [Li
+] within the leachate may be influenced by two main factors. During bioleaching, EPSs are secreted by microorganisms during growth. These entities are typically composed of charged functional groups or macromolecular polymers, such as polysaccharides, proteins, and nucleic acids, which may engage in electrostatic interactions or complexation with Li
+. In addition, for eutrophic groups, under conditions of high ORP and [Fe
3+], iron-containing secondary minerals (such as jarosite) adsorb Li
+ or undergo Li
+ substitution to embed it into the lattice (Equation (6)).
where M is a univalent cation, such as K
+, NH
4+, H
3O
+, or Li
+.
3.3. Results of Hydrochloric Acid Treatment of Leaching Residue
To elucidate the actual leaching rate of lithium and the leaching mechanism of lepidolite, the bioleaching residues were further subjected to acid leaching treatment with a hydrochloric acid solution. After the bioleaching residue was leached with dilute hydrochloric acid for 24 h, [Li
+] was found to be highest in the eutrophic bioleaching group, followed by the oligotrophic group and, finally, the sterile control group. The leaching of lithium ions in the sterile control groups was attributed primarily to the chemical effects of dilute hydrochloric acid, resulting in a relatively low dissolved lithium content during this process. Through thermal extraction, high-temperature treatment was employed to disrupt the cell membranes of acidophilic microorganisms, thereby facilitating the release of EPSs and subsequent isolation of polysaccharides. The extracted polysaccharide derivatives demonstrated a distinct chromogenic reaction with phenol under strongly acidic conditions, yielding characteristic orange–yellow compounds (
Figure S2). Spectral analysis revealed a prominent absorbance peak at 490 nm for these compounds, with detailed quantitative data provided in
Table S2. ICP-OES analysis confirmed the presence of lithium ions in the EPS-containing supernatant (
Table S3), indicating that the lithium ions released in the oligotrophic bioleaching system were also attributed to the dissociation of EPSs under the action of dilute hydrochloric acid, although the EPS content was very small. For the eutrophic bioleaching groups, the release of lithium ions included the chemical effects of dilute hydrochloric acid, EPS dissociation, and the release of Li
+ adsorbed by secondary minerals. A comprehensive comparison among these three groups revealed significant differences in lithium leaching after hydrochloric acid treatment, indicating that during bioleaching, most of the Li
+ existed in a dissolved state in the solution, whereas only a small amount was adsorbed by EPSs and secondary minerals. This discovery provides an important basis for our in-depth understanding of the migration and transformation mechanism of Li
+. Notably, the results in
Figure 7 also show that the leaching rates of lithium for all the groups were positively correlated with the mechanical activation time. The leaching residue activated for 150 min had the best leaching effect with dilute hydrochloric acid, and the lithium concentrations of the solutions were 62.33 mg/L, 79.40 mg/L, and 91.13 mg/L. Therefore, to further study the role of the microbial community in bioleaching and the relevant mechanism, the groups with lepidolite activated for 150 min and nonactivated lepidolite were selected for subsequent analyses.
3.5. Analysis of the Microbial Community Structure
Compared with bioleaching with a single bacterium, bioleaching with a microbial community has more advantages, and the interaction between microbial populations is highly important for improving the efficiency of ore leaching [
24]. Variations in chemical and physical conditions during the bioleaching process, such as the pH and the Fe
3+/Fe
2+ ratio, are the primary factors that influence microbial dynamics [
25]. Analysis of the composition and structural changes in the microbial communities of the eutrophic and oligotrophic groups (nonactivated/activated for 150 min) after 14 days of bioleaching was conducted (
Figure 10). Before mechanical activation, the oligotrophic groups were dominated by Firmicutes, with an abundance of 54.4% (
Figure 10a). Compared with the abundances before bioleaching, the the abundances of Proteobacteria and Nitrospirota decreased by 37.6% and 41.4%, respectively. Notably, Nitrospirota constituted a mere 0.38% of the total microbial population. In contrast, the eutrophic groups were primarily composed of Proteobacteria (51.4%) and Nitrospirota (41.8%). This might be because environments rich in metal ions provide suitable conditions for the growth of Proteobacteria. Moreover, Nitrospirota utilizes pyrite as an energy source and is acid tolerant; thus, it has a competitive advantage in eutrophic leaching groups, leading to an increase in abundance. On the other hand, some microorganisms belonging to the Firmicutes phylum are sensitive to acidic environments, which makes oligotrophic conditions more favorable for the growth of microorganisms within the Firmicutes phylum. Mechanical activation significantly increased the diversity of microbial communities at the phylum level. After 150 min of activation, significant changes occurred in the proportion of microorganisms at the phylum level in the oligotrophic group. Firmicutes and Proteobacteria accounted for 21.8% and 18.7%, respectively. Although the absolute values of their proportions were relatively small, they were still dominant communities. In comparison, the composition of the microbial communities in the eutrophic group also increased, but with extremely low relative abundances. The relative abundances of Nitrospirota, Proteobacteria, and Firmicutes did not change significantly, but Proteobacteria and Nitrospirota still held absolute dominance. Overall, in both the oligotrophic and eutrophic groups, mechanical activation altered the composition of microbial communities to some extent, which may be related to the adaptability of microorganisms to environmental conditions.
According to the histogram of the relative abundance of microorganisms at the genus level (
Figure 10b), before the bioleaching experiment,
Acidithiobacillus,
Leptospirillum, and unclassified
Acetobacteraceae were absolutely dominant. Under oligotrophic conditions, there was a great difference in the diversity of microbial genera before and after leaching, and other acidophilic bacteria (others) were absolutely dominant; however, owing to the lack of sufficient energy substrates such as Fe(II) and FeS
2, the relative abundances of
Acidithiobacillus and
Leptospirillium decreased sharply. The increase in the content of
Alicyclobacillus to 28% may be because
Alicyclobacillus can metabolize organic matter produced by other microorganisms; in addition, its own metabolites can promote the leaching of nonsulfide ores, such as silicate ores, through complexation, reduction, acid hydrolysis, and other mechanisms [
26]. The effect of pyrite on the leaching bacterial community was more obvious [
27].
Acidithiobacillus and
Leptospirillium were the dominant species after leaching under eutrophic conditions, and the relative abundance of
Sulfobacillus also increased. Mechanical activation also affects microbial communities at the genus level in both oligotrophic and eutrophic groups. Mechanical activation resulted in the continuous dominance of unclassified bacteria in the oligotrophic groups, a significant decrease in the abundance of
Alicyclobacillus, and the emergence of new genera such as
Vibrio and
Chloroflexi with certain abundances after activation. In comparison, in the eutrophic groups,
Leptospirillum and
Acidithiobacillus remained the dominant genera both before and after activation. Moreover, there was an increase in
Sulfobacillus abundance. Furthermore, some new genera appeared for the first time after activation but with relatively low abundances. These results suggest that mechanical activation seems to have a certain reshaping effect on the microbial community structure. The mechanical stress imposed by the activation process likely disrupts the existing microbial equilibrium, favoring the growth of certain taxa and suppressing others. Such perturbations in microbial communities can potentially affect various ecosystem functions and processes.
A heatmap diagram was generated to visualize the microbial sequencing data before and after leaching. According to the gradient and similarity of color, the differences in community composition and species and the similarities of microorganisms at the taxonomic level before and after leaching were determined (
Figure 10c) [
28]. When the lepidolite was not activated, the relative abundances of unclassified
Leptospirillum,
Acidithiobacillus caldus, and
Acidisphaera sp. PS110 decreased significantly after leaching in the oligotrophic groups, but the opposite trend was observed in the eutrophic groups. The changes in oligotrophic and eutrophic microorganisms before and after mechanical activation were similar at the species level. Notably, after 150 min of mechanical activation, the relative abundance of
Sulfobacillus thermosulfidooxidans significantly increased in the eutrophic leaching groups. These results indicate that for the bioleaching processes of lepidolite, mechanical activation increases the diversity of the microbial community, which helps to improve the overall function of the microbial community, enhances adaptability to different environmental conditions, increases the overall metabolic efficiency, and thus increases the leaching rate of ions.
3.6. A Discussion of the Leaching Rate and Leaching Mechanism
In this study, it was found that the reaction activity of ions in lepidolite can be greatly improved by mechanochemical activation combined with interactions between microorganisms and minerals. This could be attributed to the fact that after mechanical activation, microorganisms are more likely to penetrate into the depths of these lepidolite craters and cracks and continue to erode the ore [
29]. Generally, the recovery of lithium is mainly concentrated in the leaching solution. After 14 days of leaching, the leaching rates of lithium in the sterile control group and oligotrophic and eutrophic bioleaching groups after activation for 150 min were 11.3%, 20.8%, and 24.9%, respectively, which were 10.53%, 17.89%, and 20.04% greater than those without activation (
Figure 11). The leaching rates of aluminum ions before and after mechanical activation are presented in
Figure S5. In addition, regardless of whether the lepidolite was activated, eutrophic bioleaching improved the leaching rate of the elements in the lepidolite. Notably, under the experimental conditions of mechanical activation, although the leaching rate was still relatively low, the leaching rate of lithium under the experimental conditions in this study significantly improved compared with that in the literature, approximately 3 times greater than that reported by Sedlakova-Kadukova et al. [
7], and the leaching time (14 d) was shorter than that (41 d), indicating great application potential. Moreover, Kirk et al. [
30] recently reported that the bioleaching efficiency of lithium from lepidolite using
Acidiothiobacillus ferrooxidans was only 14% within 30 days. In addition, the current mainstream lithium extraction technology for lepidolite is the sulfate roasting method. Its advantages are a relatively high recovery rate (approximately 75%) and the ability to process low-grade lepidolite. The disadvantages are the high cost of the raw material potassium sulfate, long process flow, relatively large amount of slag, and higher comprehensive costs than those of salt lakes and spodumene, which are approximately CNY 60,000–80,000 per ton in China. In this work, the bioleaching method eliminated the need to consider issues such as waste residue treatment, equipment corrosion and maintenance, requirements for other materials, and subsequent environmental remediation. This seemingly results in substantial cost savings. Compared with processes involving strong acids, strong alkalis, and high-temperature roasting, this method reduces both energy consumption and costs while being environmentally friendly. Furthermore, although the bioleaching cycle is relatively long, employing mechanical activation as a pretreatment effectively enhances the extraction efficiency of elements from lepidolite.
The mechanical activation pretreatment process can enhance the disruption of Si-O-K and Si-O-Li structures within the lepidolite laminar structure, thereby reducing the bond strength of Li-O [
11]. Meanwhile, the average particle size of lepidolite is significantly reduced, and the specific surface area is increased. Moreover, during the subsequent bioleaching process, more cracks and pores are generated in lepidolite, which enlarges the contact area between microorganisms and minerals, providing more favorable conditions for growth and metabolic activities and thus effectively increasing the bioleaching efficiency. Additionally, bioleaching under mechanical activation increases the diversity of the microbial community, thereby positively affecting the increase in the lithium leaching rate. On the basis of the above analysis, a schematic diagram of the leaching process of lepidolite with different interaction groups was proposed, as shown in
Figure 12. The conventional leaching of Li generally uses a large amount of protonic acid to enhance the decomposition of silicon–oxygen tetrahedrons in lamellar structures, resulting in the dislocation of stable Si-O-Li structures, increased Li-O activity, and efficient leaching of Li between lamellar structures [
11]. Regardless of whether mechanical activation is performed before leaching, owing to the initial acidic environment, different leaching groups can slowly promote leaching. The presence of microorganisms significantly promotes the leaching of elements from lepidolite. On the one hand, silicate bacteria and autotrophic bacteria in the microbial community may adsorb onto the surface of lepidolite, playing a direct role in leaching. On the other hand, the extracellular polymeric substances produced by microorganisms can adsorb positively charged lithium ions through electrostatic or complexation interactions. Although there is no pyrite energy substrate in oligotrophic groups, microorganisms are forced to obtain nutrients needed for life activities directly from lepidolite, thus promoting leaching; in eutrophic bioleaching groups, acidophilic microorganisms oxidize pyrite, and the subsequent hydrolysis of Fe
3+ in the solution releases H
+. The protons enhance the decomposition of silicon–oxygen tetrahedrons in lamellar structures, resulting in the dislocation of stable Si-O-Li structures, increased Li-O activity, and the efficient leaching of Li between lamellar structures [
11], and separating metal ions from ore particles by reducing their binding strength, enhancing the mobility of metal ions and promoting their leaching from minerals. Additionally, secondary minerals form in eutrophic bioleaching groups because their high iron content can also adsorb lithium ions. The adsorption of lithium by cells and secondary minerals greatly contributes to the dissolution of lepidolite, thus improving the leaching efficiency of lithium. This is due to the rapid accumulation of lithium ions released in the solution on the cell surface and in secondary minerals, which can cause the dissolution equilibrium of lepidolites to shift in the direction of Li
+ ion release. Consequently, in this study, pretreatment of lepidolite with mechanical activation prior to leaching effectively improved the leaching efficiency of the tested elements.