Differential Mechanisms of Microbial As(III) and Sb(III) Oxidation and Their Contribution to Tailings Reclamation.

Mine tailings are extremely oligotrophic environments frequently contaminated with elevated As and Sb, making As(III) and Sb(III) oxidation potentially important energy sources for the tailing microbiome. Although they have been proposed to share similar metabolic pathways, a systemic comparison of the As(III) and Sb(III) oxidation mechanisms and energy utilization efficiencies requires further elucidation. In this study, we employed a combination of physicochemical, molecular, and bioinformatic analyses to compare the kinetic and genetic mechanisms of As(III) and Sb(III) oxidation as well as their respective energy efficiencies for fueling the key nutrient acquisition metabolisms. Thiobacillus and Rhizobium spp. were identified as functional populations for both As(III) and Sb(III) oxidation in mine tailings by DNA-stable isotope probing. However, these microorganisms mediated As(III) and Sb(III) oxidation via different metabolic pathways, resulting in preferential oxidation of Sb(III) over As(III). Notably, both As(III) and Sb(III) oxidation can facilitate nitrogen fixation and phosphate solubilization in mine tailings, with Sb(III) oxidation being more efficient in powering these processes. Thus, this study provided novel insights into the microbial As(III) and Sb(III) oxidation mechanisms and their respective nutrient acquisition efficiencies, which may be critical for the reclamation of mine tailings.

Microbial Sulfur and Arsenic Oxidation Facilitate the Establishment of Biocrusts during Reclamation of Degraded Mine Tailings.

Degraded tailings generated by the mining of metal ores are major environmental threats to the surrounding ecosystems. Tailing reclamation, however, is often impeded due to adverse environmental conditions, with depleted key nutrients (i.e., nitrogen (N) and phosphorus (P)) and elevated sulfur and metal(loid) concentrations. Formation of biocrusts may significantly accelerate nutrient accumulation and is therefore an essential stage for tailing reclamation. Although suggested to play an important role, the microbial community composition and key metabolisms in biocrusts remain largely unknown and are therefore investigated in the current study. The results suggested that sulfur and arsenic oxidation are potential energy sources utilized by members of predominant biocrust bacterial families, including Beijerinckiaceae, Burkholderiaceae, Hyphomicrobiaceae, and Rhizobiaceae. Accordingly, the S and As oxidation potentials are elevated in biocrusts compared to those in their adjacent tailings. Biocrust growth, as proxied by chlorophyll concentrations, is enhanced in treatments supplemented with S and As. The elevated biocrust growth might benefit from nutrient acquisition services (i.e., nitrogen fixation and phosphorus solubilization) fueled by microbial sulfur and arsenic oxidation. The current study suggests that sulfur- and arsenic-oxidizing microorganisms may play important ecological roles in promoting biocrust formation and facilitating tailing reclamation.

Mycobacteriaceae Mineralizes Micropolyethylene in Riverine Ecosystems.

Microplastic (MP) contamination is a serious global environmental problem. Plastic contamination has attracted extensive attention during the past decades. While physiochemical weathering may influence the properties of MPs, biodegradation by microorganisms could ultimately mineralize plastics into CO2. Compared to the well-studied marine ecosystems, the MP biodegradation process in riverine ecosystems, however, is less understood. The current study focuses on the MP biodegradation in one of the world's most plastic contaminated rivers, Pearl River, using micropolyethylene (mPE) as a model substrate. Mineralization of 13C-labeled mPE into 13CO2 provided direct evidence of mPE biodegradation by indigenous microorganisms. Several Actinobacteriota genera were identified as putative mPE degraders. Furthermore, two Mycobacteriaceae isolates related to the putative mPE degraders, Mycobacterium sp. mPE3 and Nocardia sp. mPE12, were retrieved, and their ability to mineralize 13C-mPE into 13CO2 was confirmed. Pangenomic analysis reveals that the genes related to the proposed mPE biodegradation pathway are shared by members of Mycobacteriaceae. While both Mycobacterium and Nocardia are known for their pathogenicity, these populations on the plastisphere in this study were likely nonpathogenic as they lacked virulence factors. The current study provided direct evidence for MP mineralization by indigenous biodegraders and predicted their biodegradation pathway, which may be harnessed to improve bioremediation of MPs in urban rivers.

VV Reduction by Polaromonas spp. in Vanadium Mine Tailings.

Vanadium (V) is an important metal with critical industrial and medical applications. Elevated V contamination, however, can be a threat to the environment and human health. Microorganisms can reduce the more toxic and mobile VV to the less toxic and immobile VIV, which could be a detoxification and energy metabolism strategy adopted by V-reducing bacteria (VRB). The limited understanding of microbial responses to V contamination and the mechanisms for VV reduction, however, hamper our capability to attenuate V contamination. This study focused on determining the microbial responses to elevated V concentration and the mechanisms of VV reduction in V tailings. The bacterial communities were characterized and compared between the V tailings and the less contaminated adjacent mineral soils. Further, VV-reducing enrichments indicated that bacteria associated with Polaromonas, a genus belonging to the family Burkholderiaceae, were potentially responsible for VV reduction. Retrieved metagenome-assembled genomes (MAGs) suggested that the Polaromonas spp. encoded genes (cymA, omcA, and narG) were responsible for VV reduction. Additionally, Polaromonas spp. was metabolically versatile and could use both organic and inorganic electron donors. The metabolic versatility of Polaromonas spp. may be important for its ability to flourish in the V tailings.

Investigation of the Ecological Roles of Putative Keystone Taxa during Tailing Revegetation.

Metal contamination released from tailings is a global environmental concern. Although phytoremediation is a promising remediation method, its practice is often impeded by the adverse tailing geochemical conditions, which suppress biological activities. The ecosystem services provided by indigenous microorganisms could alter environmental conditions and facilitate revegetation in tailings. During the process, the keystone taxa of the microbial community are assumed an essential role in regulating the community composition and functions. The identity and the environmental functions of the keystone taxa during tailing revegetation, however, remain unelucidated. The current study compared the microbial community composition and interactions of two contrasting stibnite (Sb2S3) tailings, one revegetated and one unvegetated. The microbial interaction networks and keystone taxa were significantly different in the two tailings. Similar keystone taxa were also identified in other revegetated tailings, but not in their corresponding unvegetated tailings. Metagenome-assembled genomes (MAGs) indicated that the keystone taxa in the revegetated tailing may use both organic and inorganic energy sources (e.g., sulfur, arsenic, and antimony). They could also facilitate plant growth since a number of plant-growth-promoting genes, including phosphorus solubilization and siderophore production genes, were encoded. The current study suggests that keystone taxa may play important roles in tailing revegetation by providing nutrients, such as P and Fe, and promoting plant growth.

Chemolithoautotropic Diazotrophy Dominates the Nitrogen Fixation Process in Mine Tailings.

Nutrient deficiency, especially bio-available nitrogen deficiency, often impedes the bioremediation efforts of mining generated tailings. Biological nitrogen fixation is a critical process necessary for the initial nitrogen buildup in tailings. Current knowledge regarding the diazotrophs that inhabit tailings is still in its infancy. Therefore, in this study, a comprehensive investigation combining geochemical characterization, sequence analyses, molecular techniques, and activity measurements was conducted to characterize the diazotrophic community residing in tailing environments. Significant differences between tailings and their adjacent soils in prokaryotic and diazotrophic communities were detected. Meanwhile, strong and significant correlations between the absolute abundance of the nitrogen fixation (nifH), carbon fixation (cbbL), sulfur oxidation (soxB), and arsenite oxidation (aioA) genes were observed in the tailings but not in the soils. The reconstructed nif-containing metagenome-assembled genomes (MAGs) suggest that the carbon fixation and sulfur oxidation pathways were important for potential diazotrophs inhabiting the tailings. Activity measurements further confirmed that diazotrophs inhabiting tailings preferentially use inorganic electron donors (e.g., elemental sulfur) compared to organic electron donors (e.g., sucrose), while diazotrophs inhabiting soils preferred organic carbon sources. Collectively, these findings suggest that chemolithoautotrophic diazotrophs may play essential roles in acquiring nutrients and facilitating ecological succession in tailings.

Bacteria associated with Comamonadaceae are key arsenite oxidizer associated with Pteris vittata root.

Pteris vittata (P. vittata), an arsenic (As) hyperaccumulator commonly used in the phytoremediation of As-contaminated soils, contains root-associated bacteria (RAB) including those that colonize the root rhizosphere and endosphere, which can adapt to As contamination and improve plant health. As(III)-oxidizing RAB can convert the more toxic arsenite (As(III)) to less toxic arsenate (As(V)) under As-rich conditions, which may promote plant survial. Previous studies have shown that microbial As(III) oxidation occurs in the rhizospheres and endospheres of P. vittata. However, knowledge of RAB of P. vittata responsible for As(III) oxidation remained limited. In this study, members of the Comamonadaceae family were identified as putative As(III) oxidizers, and the core microbiome associated with P. vittata roots using DNA-stable isotope probing (SIP), amplicon sequencing and metagenomic analysis. Metagenomic binning revealed that metagenome assembled genomes (MAGs) associated with Comamonadaceae contained several functional genes related to carbon fixation, arsenic resistance, plant growth promotion and bacterial colonization. As(III) oxidation and plant growth promotion may be key features of RAB in promoting P. vittata growth. These results extend the current knowledge of the diversity of As(III)-oxidizing RAB and provide new insights into improving the efficiency of arsenic phytoremediation.

Microbially mediated sulfur oxidation coupled with arsenate reduction within oligotrophic mining-impacted habitats.

Arsenate [As(V)] reduction is a major cause of arsenic (As) release from soils, which threatens more than 200 million people worldwide. While heterotrophic As(V) reduction has been investigated extensively, the mechanism of chemolithotrophic As(V) reduction is less studied. Since As is frequently found as a sulfidic mineral in the environment, microbial mediated sulfur oxidation coupled to As(V) reduction (SOAsR), a chemolithotrophic process, may be more favorable in sites impacted by oligotrophic mining (e.g. As-contaminated mine tailings). While SOAsR is thermodynamically favorable, knowledge regarding this biogeochemical process is still limited. The current study suggested that SOAsR was a more prevalent process than heterotrophic As(V) reduction in oligotrophic sites, such as mine tailings. The water-soluble reduced sulfur concentration was predicted to be one of the major geochemical parameters that had a substantial impact on SOAsR potentials. A combination of DNA stable isotope probing and metagenome binning revealed members of the genera Sulfuricella, Ramlibacter, and Sulfuritalea as sulfur oxidizing As(V)-reducing bacteria (SOAsRB) in mine tailings. Genome mining further expanded the list of potential SOAsRB to diverse phylogenetic lineages such as members associated with Burkholderiaceae and Rhodocyclaceae. Metagenome analysis using multiple tailing samples across southern China confirmed that the putative SOAsRB were the dominant As(V) reducers in these sites. Together, the current findings expand our knowledge regarding the chemolithotrophic As(V) reduction process, which may be harnessed to facilitate future remediation practices in mine tailings.

Arsenic (As) oxidation by core endosphere microbiome mediates As speciation in Pteris vittata roots.

Pteris vittata is an arsenic(As)-hyperaccumulator that may be employed in phytoremediation of As-contaminated soils. P. vittata-associated microbiome are adapted to elevated As and may be important for host survival under stresses. Although P. vittata root endophytes could be critical for As biotransformation in planta, their compositions and metabolisms remain elusive. The current study aims to characterize the root endophytic community composition and As-metabolizing potentials in P. vittata. High As(III) oxidase gene abundances and rapid As(III) oxidation activity indicated that As(III) oxidation was the dominant microbial As-biotransformation processes compared to As reduction and methylization in P. vittata roots. Members of Rhizobiales were the core microbiome and the dominant As(III) oxidizers in P. vittata roots. Acquasition of As-metabolising genes, including both As(III) oxidase and As(V) detoxification reductase genes, through horizontal gene transfer was identified in a Saccharimonadaceae genomic assembly, which was another abundant population residing in P. vittata roots. Acquisition of these genes might improve the fitness of Saccharimonadaceae population to elevated As concentrations in P. vittata. Diverse plant growth promoting traits were encoded by the core root microbiome populations Rhizobiales. We propose that microbial As(III) oxidation and plant growth promotion are critical traits for P. vittata survival in hostile As-contaiminated sites.

Desulfurivibrio spp. mediate sulfur-oxidation coupled to Sb(V) reduction, a novel biogeochemical process.

Antimony (Sb) contamination released from mine tailings represents a global threat to natural ecosystems and human health. The geochemical conditions of Sb tailings, which are oligotrophic and replete in sulfur (S) and Sb, may promote the coupled metabolism of Sb and S. In this study, multiple lines of evidence indicate that a novel biogeochemical process, S oxidation coupled to Sb(V) reduction, is enzymatically mediated by Desulfurivibrio spp. The distribution of Desulfurivibrio covaried with S and Sb concentrations, showing a high relative abundance in Sb mine tailings but not in samples from surrounding sites (i.e., soils, paddies, and river sediments). Further, the metabolic potential to couple S oxidation to Sb(V) reduction, encoded by a non-canonical, oxidative sulfite reductase (dsr) and arsenate reductase (arrA) or antimonate reductase (anrA), respectively, was found to be common in Desulfurivibrio genomes retrieved from metal-contaminated sites in southern China. Elucidation of enzymatically-catalyzed S oxidation coupled to Sb(V) reduction expands the fundamental understanding of Sb biogeochemical cycling, which may be harnessed to improve remediation strategies for Sb mine tailings.

Investigation of the antimony fractions and indigenous microbiota in aerobic and anaerobic rice paddies.

The accumulation of antimony (Sb) by rice is a severe threat to exposed populations. Previous studies demonstrated that, compared to flooded (anaerobic) water management, dry cultivation management (aerobic) could substantially decrease As, an analog of Sb, uptake by rice. However, the effects of different water management strategies on the accumulation of Sb by rice are less understood. It is proposed that microorganisms play an important role in regulating Sb mobility in rice paddies. Hence, the current study compared the microbial communities in rice paddies receiving different water management, i.e., flooded (anaerobic) and dry (aerobic)) rice cultivation. Significant decrease in Sb uptake by rice, in both the roots and grains, was observed under the aerobic compared to the anaerobic conditions. This could partially be attributed to the differences in the microbial communities as shaped by the redox environment. In aerobic soils, the gene responsible for Sb oxidation (i.e., aioA) was significantly, while in anaerobic soils the gene responsible for Sb reduction (i.e., arrA) was enriched, suggesting that variation in redox conditions may trigger different microbial responses. Accordingly, geochemical analysis indicated that accumulation of Sb(III) was only observed under anaerobic conditions, but not under aerobic conditions. The environment-microbe interactions were distinct between the two treatments with a greater number of interactions between Sb fractions and the microbial assemblage under anaerobic conditions, while Eh was the most influential geochemical parameter under aerobic conditions. Finally, the presence of a core microbiome under the two conditions suggested the possibility of microorganisms that support rice growth, nutrition, and health. The reduction of Sb in rice grain significantly decreases Sb exposure to the residents in Sb contaminated regions, and should be considered for future rice cultivation practices.

Arsenic and antimony co-contamination influences on soil microbial community composition and functions: Relevance to arsenic resistance and carbon, nitrogen, and sulfur cycling.

Microorganisms can mediate arsenic (As) and antimony (Sb) transformation and thus change the As and Sb toxicity and mobility. The influence of As and Sb on the innate microbiome has been extensively characterized. However, how microbial metabolic potentials are influenced by the As and Sb co-contamination is still ambiguous. In this study, we selected two contrasting sites located in the Shimen realgar mine, the largest realgar mine in Asia, to explore the adaptability and response of the soil microbiome to As and Sb co-contamination and the impact of co-contamination on microbial metabolic potentials. It is observed that the geochemical parameters, including the As and Sb fractions, were the driving forces that reshaped the community composition and metabolic potentials. Bacteria associated with Bradyrhizobium, Nocardioides, Sphingomonas, Burkholderia, and Streptomyces were predicted to be tolerant to high concentrations of As and Sb. Co-occurrence network analysis revealed that the genes related to C fixation, nitrate/nitrite reduction, N fixation, and sulfate reduction were positively correlated with the As and Sb fractions, suggesting that As and Sb biogeochemical cycling may interact with and benefit from C, N, and S cycling. The results suggest that As and Sb co-contamination not only influences As-related genes, but also influences other genes correlated with microbial C, N, and S cycling.

Citric acid and AMF inoculation combination-assisted phytoextraction of vanadium (V) by Medicago sativa in V mining contaminated soil.

The use of citric acid (CA) chelator to facilitate metal bioavailability is a promising approach for the phytoextraction of heavy metal contaminants. However, the role of the CA chelator associated with arbuscular mycorrhizal fungi (AMF) inoculation on phytoextraction of vanadium (V) has not been studied. Therefore, in this study, a greenhouse pot experiment was conducted to evaluate the combined effect of CA chelator and AMF inoculation on growth performance and V phytoextraction of plants in V-contaminated soil. The experiment was performed via CA (at 0, 5, and 10 mM kg-1 soil levels) application alone or in combination with AMF inoculation by Medicago sativa Linn. (M. sativa). Plant biomass, root mycorrhizal colonization, P and V accumulation, antioxidant enzyme activity in plants, and soil chemical speciation of V were evaluated. Results depicted (1) a marked decline in plant biomass and root mycorrhizal colonization in 5- and 10-mM CA treatments which were accompanied by a significant increased V accumulation in plant tissues. The effects could be attributed to the enhanced acid-soluble V fraction transferring from the reducible fraction. (2) The presence of CA significantly enhanced P acquisition while the P/V concentration ratio in plant shoots and roots decreased, owing to the increased V translocation from soil to plant. (3) In both CA-treated soil, AMF-plant symbiosis significantly improved dry weight (31.4-73.3%) and P content (37.3-122.5%) in shoots and roots of M. sativa. The combined treatments also showed markedly contribution in reduction of malondialdehyde (MDA) content (12.8-16.2%) and higher antioxidants (SOD, POD, and CAT) activities in the leaves. This suggests their combination could promote growth performance and stimulate antioxidant response to alleviate V stress induced by CA chelator. (4) Taken together, 10 mM kg-1 CA application and AMF inoculation combination exhibited a higher amount of extracted V both in plant shoots and roots. Thus, citric acid-AMF-plant symbiosis provides a novel remediation strategy for in situ V phytoextraction by M. sativa in V-contaminated soil.

Stable-isotope probing coupled with high-throughput sequencing reveals bacterial taxa capable of degrading aniline at three contaminated sites with contrasting pH.

The biodegradation of aniline is an important process related to the attenuation of aniline pollution at contaminated sites. Aniline contamination could occur in various pH (i.e., acidic, neutral, and alkaline) environments. However, little is known about preferred pH conditions of diverse aniline degraders at different sites. This study investigated the active aniline degraders present under contrasting pH environments using three aniline-contaminated cultures, namely, acidic sludge (ACID-S, pH 3.1), neutral river sediment (NEUS, pH 6.6), and alkaline paddy soil (ALKP, pH 8.7). Here, DNA-based stable isotope probing coupled with high-throughput sequencing revealed that aniline degradation was associated with Armatimonadetes sp., Tepidisphaerales sp., and Rhizobiaceae sp. in ACID-S; Thauera sp., Zoogloea sp., and Acidovorax sp. in NEUS; Delftia sp., Thauera sp., and Nocardioides sp. in ALKP. All the putative aniline-degrading bacteria identified were present in the "core" microbiome of these three cultures; however, only an appropriate pH may facilitate their ability to metabolize aniline. In addition, the biotic interactions between putative aniline-degrading bacteria and non-direct degraders showed different characteristics in three cultures, suggesting aniline-degrading bacteria employ diverse survival strategies in different pH environments. These findings expand our current knowledge regarding the diversity of aniline degraders and the environments they inhabit, and provide guidance related to the bioremediation of aniline contaminated sites with complex pH environments.

Solar-driven, self-sustainable electrolysis for treating eutrophic river water: Intensified nutrient removal and reshaped microbial communities.

River ecosystems are the most important resource of surface freshwater, but they have frequently been contaminated by excessive nutrient input of nitrogen (N) and phosphorus (P) in particular. An efficient and economic river water treatment technology that possesses the capacity of simultaneous N and P removal is urgently required. In this study, a solar-driven, self-sustainable electrolytic treatment was conducted in situ to intensify N and P removal from eutrophic river water. Solar panel was applied to provide the electrolysis setups with energy (voltage 10 ± 0.5 V), and the current density was controlled to be 0.06 ± 0.02 mA cm-2. Results indicated that the average removal efficiencies of total N (TN) and total P (TP) under electrolysis conditions reached 72.4 ± 11.7 and 13.8 ± 5.3 mg m-2 d-1, which were 3.7- and 4.7-fold higher compared to untreated conditions. Enhanced TN removal mainly reflected the abatement of nitrate N (NO3--N) (80.6 ± 4.1%). The formation of ferric ions through the electro-dissolution of the sacrificial iron anode improved TP removal by coprecipitation with SPS. Combined high-throughput sequencing and statistical analyses revealed that electrolysis significantly reshaped the microbial communities in both the sediment-water interface and suspended sediment (SPS), and hydrogenotrophic denitrifiers (e.g., Hydrogenophaga) were highly enriched under electrolysis conditions. These findings indicated that in situ electrolysis is a feasible and effective technology for intensified nutrient removal from river water.

Comparative characterization of microbial communities that inhabit arsenic-rich and antimony-rich contaminated sites: Responses to two different contamination conditions.

Due to extensive mining and industrial activities, arsenic (As) and antimony (Sb) contaminations are becoming a global environmental concern. Both As and Sb are toxic and carcinogenic metalloids from the group 15 in the periodic table. Since As and Sb share many similar geochemical properties, it is often assumed that they exert similar environmental pressure on the native microbial communities. This hypothesis, however, still requires further confirmation. In the current study, a systematic comparison of microbial responses to As and Sb contamination were conducted. The results suggested that regular geochemical parameters, such as pH, nitrate, and TOC, were the driving forces for shaping the microbial community. In correspondence, two heavily contaminated groups showed similar microbial community compositions and the same microbial populations were enriched. The interactions between the contaminant fractions (As and Sb related fractions) and the individual OTUs, however, suggested the different and more diverse impacts of As comparing to Sb fractions, with more taxa significantly impacted by As species comparing to Sb species. The identification of the keystone taxa in the heavily contaminated samples revealed a group of microbial populations that could survive in both As and Sb heavily contaminated conditions and may providing critical environmental services to the community. Further investigation of these key microbial populations may provide valuable insights on employing these microorganisms for remediation applications.

Bacterial response to sharp geochemical gradients caused by acid mine drainage intrusion in a terrace: Relevance of C, N, and S cycling and metal resistance.

A unique terrace with sharp gradient of environmental conditions was selected to study the microbial response and survival strategies to the extreme environments introduced by acid mine drainage (AMD) contamination. A combination of geochemical analyses, metagenomic sequencing, ex-situ microcosm setups, and statistical analyses were used to investigate the environment-microbe interactions. The microbial communities and metabolic potentials along the terrace were studied by focusing on the genes associated with important biogeochemical processes (i.e., C, N, S cycling and metal resistance). Results show that the variations of geochemical parameters substantially shaped the indigenous microbial communities. Sharp environmental gradients also impacted the microbial metabolic potentials, especially for C, N, and S cycling. Although the relative abundances of carbon fixing genes did not significantly vary along the environmental gradients, the taxa for carbon fixation varied significantly in more contaminated fields versus less contaminated fields, indicating the effects of AMD contamination on the autotrophic microbial communities. AMD input also influenced the N cycling, especially for nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). In addition, ex situ experiments were undertaken to evaluate the effects of AMD contamination on nitrogen fixation rates. Random Forest (RF) analysis indicated that nitrate, pH, total N, TOC exhibited positive correlations with the rates of nitrogen fixation while total Fe, Fe(III), and sulfate showed negative effects. Two co-occurrence networks at taxonomic and genomic levels indicated that geochemical parameters such as pH, TOC, total N, total S, and total Fe substantially influenced the innate microbial communities and their metabolic potentials. The current study provides an understanding for microbial response to AMD contamination and lays the foundation for future potential AMD bioremediation.

Uncovering microbial responses to sharp geochemical gradients in a terrace contaminated by acid mine drainage.

Acid mine drainage (AMD) is harmful to the environment and human health. Microorganisms-mineral interactions are responsible for AMD generation but can also remediate AMD contamination. Understanding the microbial response to AMD irrigation will reveal microbial survival strategies and provide approaches for AMD remediation. A terrace with sharp geochemical gradients caused by AMD flooding were selected to study the microbial response to changes in environmental parameters related to AMD contamination. AMD intrusion reduced soil microbial community diversity and further changed phylogenetic clustering patterns along the terrace gradient. We observed several genera seldom reported in AMD-related environments (i.e., Corynebacterium, Ochrobactrum, Natronomonas), suggesting flexible survival strategies such as nitrogen fixation, despite the poor nutritional environment. A co-occurrence network of heavily-contaminated fields was densely connected. The phyla Proteobacteria, Acidobacteria, Chloroflexi, and Euryarchaeota were all highly interconnected members, which may affect the formation of AMD. Detailed microbial response to different soil characterizations were highlighted by random forest model. Results revealed the top three parameters influencing the microbial diversity and interactions were pH, Fe(III), and sulfate. Various acidophilic Fe- and S-metabolizing bacteria were enriched in the lower fields, which were heavily contaminated by AMD, and more neutrophiles prevailed in the less-contaminated upper fields. Many indicator species in the lower fields were identified, including Desulfosporosinus, Thermogymnomonas, Corynebacterium, Shewanella, Acidiphilium, Ochrobactrum, Leptospirillum, and Allobaculum, representing acid-tolerant bacteria community in relevant environment. The detection of one known sulfate-reducing bacteria (i.e., Desulfosporosinus) suggested that biotic sulfate reduction may occur in acidic samples, which offers multiple advantages to AMD contamination treatment. Collectively, results suggested that the geochemical gradients substantially altered the soil microbiota and enriched the relevant microorganisms adapted to the different conditions. These findings provide mechanistic insights into the effects of contamination on the soil microbiota and establish a basis for in situ AMD bioremediation strategies.