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 (nif H), 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.

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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 (Sb 2 S 3 ) 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.