Studies On Microbiol Properties And Structure Of Bacterial Communities In Wastelands Of Copper Mine Tailings During The Process Of Natural Ecological Restoration

The wastes of copper mine tailings is a pedogenetic substrate. Formation and development of ecosystem on the wastelands of copper mine tailings is a typical primary succession. Most wastelands of copper mine tailings in Tongling, China, are restored in natural way. The tailings wastelands firstly come into being cryptogamic crust after the disposal, which is dominated by algae and moss, then herbaceous vascular plants settle down and gradually form a natural plant community. The changes in physico-chemical properties, microbial characteristics, enzyme activity in wastelands during primary succession were investigated in two wastelands of copper mine tailings near Tongling, and the structure of bacterial community, free-living nitrogen-fixing bacteria included, was examined using Polymerase Chain Reaction-Denatured Gradient Gel Electrophoresis (PCR-DGGE) approach. The Yangshanchong wasteland and Tongguanshan wasteland studied were discarded for 19 a and 30 a, respectively.Soil organic matter, total nitrogen, available phosphorus and water holding increased with primary succession, while the pH showed opposite trend. The microbial biomass C and N, and activities of urease and alkaline phosphatase in surface layer increased, while metabolic quotient decreased with development of ecosystem on the wastelands of copper mine tailings.As the development of wastelands, no significant trend of bacterial diversity was found in different successional stages of the same horizon. However, A-horizon displayed higher bacterial diversity than C-horizon, which was consistent with the change of soil organic matter and total nitrogen in content. Phylogenetic analysis of 16S rDNA V3-V5 variable region gene sequences retrieved from DGGE gels indicated that there were Proteobacteria, alpha-proteobacteria, beta-proteobacteria and garma-proteobacteria included, Firmicute, Bacilli and Clostridia included, Nitrospirae, Actinobacteria, Deinococcus-Thermus and Bacteroidetes detected in wastelands of copper mine tailings. Both the accumulation of nutrients and the type of vegetation influenced the changes of bacterial diversity in naturally reclaimed wastelands of copper mine tailings.Biological nitrogen fixing is an important source of nitrogen input in the natural ecological restoration of mine wastelands. The analysis of diversity of nifH gene in tailings samples with different plant communities from wastelands using PCR-DGGE demonstrated that the diversity of nifH gene in Yangshanchong wasteland did not display a consistent successional tendency with development of plant communities. The nitrogen-fixing microorganism community in the upper layer of tailings in Tongguanshan wasteland showed higher Shannon-Wiener diversity index than that in Yangshanchong wasteland. Phylogenetic analysis of nifH gene sequences retrieved from the DGGE gels indicated that there were mainly two taxa of free-living nitrogen-fixing microorganisms, Proteobacteria and Cyanobacteria living in the wastelands investigated. Canonical correspondence analysis based on the relationship between band patterns of DGGE profile and physico-chemical properties of tailings samples showed that the diversity of nifH gene in different tailings samples was mainly affected by loss of ignition, water content and pH of wastelands (p< 0.05). The dominant plant species and development period of plant communities by ameliorating pH, increasing organic matter and water content affected the diversity and structure of the free-living nitrogen-fixing microorganisms in wastelands of copper mine tailings.The study on free-living nitrogen-fixing microorganism communities in rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings, including Imperata cylindrica var. major, Cynodon dactylon, Zoysia sinica, Miscanthus sinensis, Miscanthus floridulus and Hippochaete ramosissimum, indicated that, free-living nitrogen-fixing microorganisms in the bulk soil in Yangshanchong wasteland showed higher diversity indices than that in the rhizosphere of dominant plant; and the nitrogen-fixing microorganism community in the rhizosphere of Imperata cylindrica var. major in Tongguanshan wasteland showed higher Shannon-Wiener diversity index than that in Yangshanchong wasteland. The diversity of free-living nitrogen-fixing microorganism communities in rhizosphere of Imperata cylindrica var. major and Hippochaete ramosissimum increased with the plant ages while Zoysia sinica showed the opposite trend. Phylogenetic analysis indicated that nifH gene sequences retrieved from DGGE gels clustered in the Proteobacteria and Cyanobacteria. Most of nifH gene fragments sequenced were not closely related to any known cultivated nitrogen-fixing bacteria and cyanobacteria, suggesting that the nifH gene sequences in wastelands of copper mine tailings investigated are unique and may represent novel sequences of nitrogen-fixing community. Our results indicated that diversity of nifH gene sequences in rhizosphere and non-rhizosphere tailings samples was affected by development period of plant community, plant species, plant life forms and physico-chemical properties of wastelands.