Diversity of free-living nitrogen-fixing microorganisms in wastelands of copper mine tailings during the process of natural ecological restoration

Biological nitrogen fixing is an important source of nitrogen input in the natural ecological restoration of mine wastelands. The diversity of nifH genes in tailings samples under different plant communities in Yangshanchong and Tongguanshan wastelands in Tong ling, was analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach. The nitrogen-fixing microorganism community in the upper layer of tailings of Tongguanshan wasteland discarded in 1980 showed higher Shannon-Wiener diversity index than that in Yangshanchong wasteland discarded in 1991. The diversity of nifH genes in Yangshanchong wasteland of copper mine tailings did not display a consistent successional tendency with development of plant communities during the process of natural ecological restoration. Phylogenetic analysis of 25 sequences of nifH gene fragments 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, most of which were unique and uncultured. Canonical correspondence analysis (CCA) based on the relationship between band patterns of DGGE profile and physico-chemical properties of tailings samples showed that the diversity of nifH genes in different tailing samples was mainly affected by loss of ignition, water content, pH and available Zn contents of wastelands. The dominant plant species and development period of plant communities by ameliorating pH, reducing the toxicity of heavy metals, increasing organic matter and water content affected the diversity and structure of the free-living nitrogen-fixing microorganisms in wastelands of copper mine tailings.