| Mine tailing pollution is one of the environmental problems of global concern.Because of its large output and high pollution risk,mine tailing will pose a serious threat to the environment.The sulfur oxidation process under the condition of mine tailing will activate and migrate the heavy metal pollutants in mine tailing and cause harm to the surrounding environment.As an important participant in the biogeochemical cycle of sulfur,sulfur oxidizing microorganisms are the main component of sulfur oxidation in mine tailing.Therefore,it is particularly important to understand the microbial driven sulfur oxidation process of mine tailing in situ.However,the composition of sulfur oxidizing microbial community in mine tailing and its impact on in-situ environment still need to be further discussed and analyzed.To solve this problem,this study starts from two aspects:on the one hand,the environmental microorganisms in tin mine mine tailing are isolated by sulfur oxidation directional separation medium;On the other hand,stable isotope probe technology was used to identify and analyze sulfur oxidizing microorganisms in mine tailing.In addition,the species identification,phylogenetic analysis and metabolic capacity analysis of sulfur oxidizing microorganisms in mine tailing were carried out by means of high-throughput sequencing and biostatistics analysis,and the impact of these microorganisms on the in-situ environment of mine tailing was explored.The main conclusions are as follows:（1）The microorganisms in tin mine tailing soil were directionally isolated and purified,and the 16S r RNA gene and sox B gene of pure bacteria were detected.It was found that the two pure bacteria had sulfur oxidation genes,named Methylversatilis sp.LJY-1and Limnobacter sp.LJY-2respectively.Based on the validation study of sulfur oxidation,the concentration of sulfate in the experimental system added with Methylversatilis sp.LJY-1 increased from 254.78±13.63 mg/L on day 0 to 1154.07±81.83 mg/L on the 22th day.Methylversatilis sp.LJY-1 was found to be a Methylversatilis genus with sulfur oxidation function for the first time.At the same time,the in-situ environmental simulation experiment was carried out on the two purified strains with sulfur oxidation function.The results showed that the hydrogen ion concentration of the experimental group inoculated with Methylversatilis sp.LJY-1 increased significantly.The hydrogen ion concentration increased by 6.10±0.35 times and 3.18±0.21 times respectively under flooding and wetting,which confirmed the sulfur oxidation process driven by Methylversatilis sp.LJY-1,Especially under the condition of flooding,it will significantly aggravate the acidification process of mine tailing.（2）Using stable isotope probe technology,arsenic and antimony sulfide minerals and labeled substrates were added to the microcosm to construct different SIP microcosm experimental groups.By detecting the concentration of sulfate,speciation arsenic and speciation antimony in the microcosm,it is found that the concentration of sulfate in the microcosm added with sulfide minerals increases significantly.At the same time,the concentrations of pentavalent arsenic and pentavalent antimony also increased significantly,which indicates that the sulfur oxidation of microorganisms in the microcosm with sulfide minerals has become an important metabolic activity,and there may be arsenic oxidation and antimony oxidation processes.（3）The DNA of microorganisms in the microcosm was extracted at different time points.After ultra-high speed centrifugation and component recovery and separation,the 16S r RNA gene of each component was quantitatively analyzed.The results showed that the relative abundance of16S r RNA gene in the heavy layer of 13C labeling treatment group was much higher than that in the light layer,which showed that microorganisms with sulfur oxidation ability in the microcosm gradually became dominant microorganisms.The heavy and light layer components of 13C labeled DNA and the light layer components of 12C labeled DNA were selected for high-throughput sequencing.The results showed that the relative abundance of Thiobacillus in the heavy layer was significantly higher than that in the light layer,indicating that Thiobacillus is the key microorganism driving the sulfur oxidation process.（4）The results of metagenome sequencing and phylogenetic analysis show that the relative abundance of Thiobacillus in the three microcosms added with sulfide minerals is very high,reaching more than 60%,indicating that Thiobacillus may be the main microorganism in the sulfur oxidation environment.According to the analysis of the metabolic capacity of the assembled genome,Thiobacillus contains aio AB,SOX complex,reverse dsr and other genes,indicating that Thiobacillus may have the potential of sulfur oxidation and arsenic oxidation.（5）Given that Thiobacillus has been reported to be a common dominant microorganism in mine tailing,this study further conducted pan genomic analysis of Thiobacillus genome to explore whether all strains of Thiobacillus have relevant metabolic capacity.The results showed that all strains of Thiobacillus had sulfur oxidation ability,but not all strains had arsenic and antimony oxidation ability. |
