Succession Rule Of Microbial Community And Functional Prediction Of Iron-sulfur Metabolic Genes In High-sulfur Abandoned Coal Mines

Acid mine water produces for a long time even after the closure of high sulphur coal mines,which seriously pollutes the groundwater and surface water,and has become a serious threat to ecological environment of the mining area.After the abandon of coal mine,the interior environment of coal mine changed from open system to closed and semi closed system.The change of environmental conditions induced the redistribution and succession of microbial community in the inner of abandoned coal mine.In this paper,the effects of acid drainage from abandoned coal mines on the microbial community,metabolic function and the distribution of iron thiometabolizing genes in the surface environment were studied through field investigation,analysis and simulation experiments.At the same time,the evolution process of water quality and the law of microbial community succession under the special environmental conditions of abandoned coal mines are studied.The main conclusions are as follows:?1?The effects of acid mine drainage from abandoned coal mines on the microbial community structure,metabolic function and iron and sulfur metabolic genes in surfacial environments were revealed.The results showed that the extremely acidic and high-iron oligotrophic environment polluted by AMD significantly decreased the richness and diversity of bacteria,and eosinophilic iron-sulfur metabolizing bacteria such as Ferrovum,Metallibacterium and Acidithiobacillus became the dominant bacteria.The results of CCA analysis showed that the distribution of bacterial community was mainly affected by iron content when soil p H<4.The results of Tax4Fun function prediction showed that microorganisms enhance cell activity and adaptability to extreme environment by enhancing the functions of signal transduction,membrane transport,energy metabolism,nucleotide metabolism,translation,replication and repair et al.Assimilation sulfate reduction genes such as cys J,cys I,cys H,cys NC,cys D and Sat are highly expressed in microorganisms of AMD polluted environment.Microorganisms synthesize substances necessary for biological metabolism by enhancing sulfate assimilation metabolism.?2?The simulation experiment of microbial community succession in abandoned coal mine under open,semi-closed and closed conditions was carried out,and three types of coal+gangue,gangue and soil+gangue were set up.Results showed Firmicutes,Proteobacteria,Actinobacteria,Nitrospirae and Chloroflexi were the dominant phyla in abandoned coal mines simulation system,and the microbial diversity in the system increased gradually with time.Sulfobacillus,Acidiferrobacter,Bacteriovoracaceae and Acidimicrobiales were the dominant genera in the simulated system,in which the relative abundance of Sulfobacillus in the gangue system was significantly higher than other groups,and the relative abundance of Acidiferrobacter in the closed/semi-closed system was significantly higher than that in the open system.?3?The 340-day simulation experiment of coal+gangue under closed conditions showed that the simulation system had the ability of continuous acid production under closed conditions,and the microbial community structure changed significantly in the process of acidizing.In the simulation system,the abundance of Firmicutes gradually decreased with time,while the abundance of Actinobacteria,Chloroflexi and Nitrospirae increased,and the abundance of Proteobacteria increased first and then decreased.At the genus level,bacteria with the ability of iron and sulfur metabolism,such as Sulfobacillus,Ferrimicrobium,Acidiferrobacter and Ferrithrix were gradually replaced by bacteria with strong tolerance to heavy metals,such as norank?o??Acidimicrobiales,norank?c??JG37-AG-4,norank?f??Family?XVII and norank?f??Bacteriovoracaceae.RDA analysis showed that the distribution of microbial community in coal+gangue system was mainly affected by SO₄^2-,p H and Mn.?4?In the open and semi-closed gangue simulation system,the abundance of Firmicutes decreased with time,while that of Proteobacteria and Actinobacteria increased.At the genus level,the heterotrophic iron-sulfur metabolizing bacteria Sulfobacillus and Ferrimicrobium in the simulated system were gradually replaced by norank?o??Acidimicrobiales and norank?f??Bacteriovoracaceae,while the abundance of autotrophic iron-sulfur oxidizing bacteria Acidiferrobacter did not change with time.RDA analysis showed that the distribution of microbial community in gangue system was mainly affected by heavy metals such as Al,Cr,Mn,Zn and so on.?5?The acid production process of abandoned coal mine simulation system under different conditions is revealed.At the initial stage of acid production,oxygen and microorganisms are the main catalytic oxidants of pyrite and Fe²⁺,and with the consumption of oxygen,Fe³⁺becomes the main oxidant of pyrite,resulting in the gradual decrease of Fe³⁺concentration and the increase of Fe²⁺concentration in the system.In the later stage,there was a significant difference in acid production between the open system and the closed system.With the gradual consumption of Fe³⁺in the closed system,the simulation system gradually reached a stable state.In the stable state,there were mainly Fe²⁺and SO₄^2-,in the water,while pyrite in the open system continued to oxidize slowly,and Fe²⁺and SO₄^2-gradually increased in the system.The acid production process of abandoned coal mine is accompanied by the dissolution of minerals and the hydrolysis of Fe³⁺.Facultative anaerobic Acidiferrobacter is the main acid-producing microorganism in the closed system.?6?The Tax4Fun function prediction method was used to reveal the change characteristics of microbial community metabolic function and iron and sulfur metabolic genes in the process of acid production in abandoned coal mines.Results showed that the metabolic functions of microorganisms such as Amino acid metabolism,Carbohydrate metabolism,Metabolism of cofactors and vitamins and Energy metabolism decreased gradually,while the metabolic functions such as Nucleotide metabolism,Glycan biosynthesis metabolism,Bacterial secretion system,Two-component system and Sulfur metabolism gradually increased.The abundance of Sulfate transport genes,Assimilatory sulfate reduction and Dissimilatory sulfate reduction and oxidation genes in the closed simulation system increased with time.There were 91 figures,24 tables and 179 references in this dissertation.