Study On The Distribution And Metabolic Potential Of Nitrogen-fixing Microbial Communities In Extreme Tailings Environments

Tailings have attracted increasing attention as they severely contaminate adjacent environments due to the large quantity are generated and the associated leaching of toxic metals.Due to adverse environmental effects,appropriate management and treatment strategies of mine tailings are urgently required.Bioremediation using plants or microorganisms is a potential method for remediating the contamination introduced by tailings.However,harsh environmental conditions,including elevated metal(loid)concentrations and nutrient deficiencies,especially bioavailable 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,which plays an important role in the environmental recovery of tailings.Current knowledge regarding the diazotrophs that inhabit tailings is still in its infancy.Therefore,in this study,a comprehensive investigation combining sequence analyses,geochemical characterization,molecular techniques,and acetylene reduction assay was conducted to characterize the diazotrophic community residing in tailing environments.The results are as follows:(1)The results of high-throughput sequencing analysis show that significant differences between tailings and their adjacent soils in prokaryotic and diazotrophic communities were detected.Less diverse prokaryotic and diazotrophic communities were established in the tailings compared to soils.Meanwhile,substantial biological differences in the prokaryotic and diazotrophic communities exist between the tailings.Within the prokaryotic communities,the main predominant bacteria in the tailings are Alphaproteobacteria,Betaproteobacteria and Gammaproteobacteria,which are significantly enriched than soil.In addition,including Rhizobiaceae,Bradyrhizobiaceae,and Burkholderiaceae,were significantly enriched in the tailings compared to soils.(2)Geochemical analysis,molecular techniques and random forest(RF)prediction analysis show that the relative abundance of nifH was negatively correlated with total organic carbon(TOC),but positively correlated with total sulfur(TS).Therefore,elevated sulfur content in the tailings may promote chemolithoautotrophic nitrogenfixation.Pearson correlation indicated that significant correlations between the absolute abundance of the nitrogen fixation(nifH),carbon fixation(cbbL)and sulfur oxidation(soxB)genes in the tailings,but not in the soils.Meanwhile,six metagenome-assembled genomes(MAGs)containing the complete nifH gene were successfully found in 136 MAGs.These MAGs all encode carbon-fixed genes,and complete SOX clusters were detected in bin41,bin57,and bin99 and some SOX clusters were detected in bin102 indicating that the carbon fixation and sulfur oxidation pathways were important for potential diazotrophs inhabiting the tailings.(3)Nitrogen fixation studies based on the acetylene reduction assay indicate that the toxic effects of arsenic and antimony on Azotobacter vinelandii are directly related to their concentration and valence state.The toxicity of arsenic and antimony to Azotobacter vinelandii increased significantly with the increase of their concentration,and Sb(?)and As(?)was more toxic than Sb(?)and As(?)on Azotobacter vinelandii.Activity measurements further confirmed that diazotrophs inhabiting tailings preferentially to use the 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.