2.1. AMD Generation and Associated Microorganisms
AMD can be rapidly generated when metal sulfide-containing materials are brought out to the Earth’s surface and exposed to moisture and air [
32]. Microorganisms can directly or indirectly utilize inorganic minerals as electron donors and irreversibly alter the physical conditions of the environments [
33,
34]. Pyrite (FeS
2) is the most abundant sulfide mineral on Earth and is an important player in the generation of mine drainage, as indicated by the following equations [
6,
35]:
Upon exposure to water and oxygen, the rate of abiotic oxidation of pyrite is low (Equation (1)), but can be greatly accelerated in the presence of certain microbes such as chemoautotrophic bacteria and archaea that mediate the oxidation of reduced iron in the pyrite (Equation (2)) [
6]. Owing to the increased concentration of Fe
3+, the pyrite is sustainably dissolved, driving the primary production of mine drainage [
21,
34]. Thiosulfate, produced by pyrite oxidation at the original stage (with high ambient pH), can then also be oxidized by some bacteria and archaea (Equation (3)) [
6]. The overall result of these reactions is an accumulation of protons, sulfate, and dissolved metals, resulting in an extreme environment for life.
During the microbial-mediated oxidation of sulfide minerals, both bacteria and archaea are involved [
36,
37]. The most extensively studied taxa of bacteria in previous studies are chemoautotrophic
Acidithiobacillus spp. and
Leptospirillum spp., which are generally recognized as the ringleader of the AMD problem [
38,
39]. Sulfur-oxidizing
Thiobacillus spp. and archaeal iron-oxidizing
Ferroplasma spp. have also been reported frequently as dominant members in some mining environments [
40,
41]. Based on the growth of autotrophs, mixotrophs, and heterotrophs,
Sulfobacillus spp. and
Acidiphilium spp. also thrive in AMD environments and influence the oxidation of pyrite, greatly increasing the diversity of microbial communities [
34,
42]. To date, the dominant lineages detected in AMD environments are
Proteobacteria,
Nitrospira,
Actinobacteria,
Firmicutes,
Acidobacteria,
Aquificae in bacteria and
Euryarchaeota,
Crenarchaeota in archaea, as summarized by Chen et al. [
22]. More than 100 known genera have been detected in extreme AMD-generating conditions in South China, revealing a considerable microbial diversity in these environments (
Figure 1). Except for several dominant species directly related to iron/sulfur-oxidizing, a large number of rare taxa constitute a “rare biosphere” in the community and may perform a crucial function in AMD ecosystems [
28,
43]. Although a large number of the observed operational taxonomic units (OTUs) cannot be classified into a specific genus, advanced high-throughput sequencing approaches offer more comprehensive information about the microbial community than traditional cultivation methods [
44,
45].
2.2. Microbial Community Function and Dynamics During AMD Generation
In AMD ecosystems, the microbial oxidation of iron and sulfur are vital geochemical processes producing Fe
3+ and SO
42−, which can be further utilized by microbes at higher trophic levels. Meanwhile, under specific environmental conditions, sulfate-reducing bacteria (SRB) and iron-reducing bacteria (IRB) can reduce Fe
3+ and SO
42−, completing the iron and sulfur cycles in such ecosystems [
46]. The nitrogen cycle is also critical for the microbial community because the resource of nitrogen is usually limited in AMD environments [
1]. However, based on a transcriptional analysis, Hua et al. found that the
nifH gene encoding enzymes involved in nitrogen fixation was highly expressed in rare taxa, suggesting that rare species likely perform essential ecological functions in AMD biogeochemistry [
28].
In generating AMD, the dissolving of sulfide minerals by associated microbes decreases the solution pH and releases a high amount of metals. To date, several attempts have been made to investigate the microbial community dynamics related to the acidification process of the mine tailings [
14,
25,
47]. Remarkable shifts in microbial community composition and structure have been found at different oxidation stages, most likely due to the distinct environmental gradients produced during AMD generation [
48,
49,
50]. For example, according to the result of a simulated experiment of oxidative dissolution of pyrite, the most dominant genera were
Tumebacillus,
Alicyclobacillus, and
Ferroplasma at the early, mid, and final stage, respectively [
47]. A detailed microbial community investigation in a natural AMD-generating environment indicated that the key species involved in different acidification stages varied greatly, and the relative abundances of most dominant lineages were significantly correlated with acidity [
25]. In many studies, archaea (mostly
Ferroplasma spp.) were found to be predominant at the late oxidation stage, which was usually characterized by low pH (<3.0) and a high concentration of metals [
47,
49,
50], revealing their favorable adaptation of harsh environmental conditions and indispensability in such ecosystems [
51]. Metagenomic as well as transcriptomic analyses deciphered significant variations in microbial community function [
27,
52,
53]. For example, previous metagenomics study of tailings acidification showed that the sulfur oxidation was mainly performed by
Thiobacillus at the early stage and by
Acidithiobacillus at the late stage [
50].
The overall alpha-diversity of the microbial community was widely observed to significantly relate to the acidification process. Liu et al. investigated the microbial communities in filed mine tailings at different acidification stages (pH range 2.0–7.0) and described a strong correlation between the pH and the diversity of microbes including the number of phylotypes and phylogenetic diversity (PD) [
25]. This gradual decrease of microbial diversity during the acidification of mine tailings was also found in other studies of natural AMD environments [
29,
48,
54], possibly owing to the stronger selective pressure on microorganisms. Chen et al. conducted simulated acidification experiments on pyrite in a laboratory to study the temporal succession of microbial communities at different oxidation stages [
47]. The microbial diversity characterized by both the community richness indices (observed OTUs, PD) and the community diversity indices (Shannon, Simpson) had no significant decrease until the conditions were below pH 3.0 (
Figure 2a–d). Particularly, the results of the Simpson index were subject to the intermediate species-richness hypothesis, which has been widely studied by ecologists about the community succession, revealing a flourish of microbes at the mid stage (
Figure 2d) [
55,
56]. In summary, microbial-mediated AMD generation results in a low alpha-diversity of microbial community at the late stage.
Although the oxidative dissolution of sulfide minerals gradually decreases the solution pH, some microbial-mediated reactions such as the chemoautotrophic sulfate reduction and nitrate reduction can consume protons and elevate the pH value, as indicated by the following equations:
Therefore, in a specific AMD ecosystem, the microbial community, pH value, and other environmental parameters fluctuate seasonally, influenced by outside environmental changes such as temperature [
10,
16]. In brief, the microbial activities in AMD markedly affect the environmental conditions, and in turn, environmental gradients over time and space shape the microbial diversity and community assembly.