Research On Microbial Community Structure Of Reclaimed Soil In Antaibao Opencast Coal Mine

Opencast coal mining caused serious environmental pollution and ecological damage.Ecological restoration of mining waste lands has become a necessary measure to maintain the stability of human living environment and sustainable development of coal industry.In this study,soil samples were collected form 16 reclamation sites(according to reclaimed vegetation and years)in reclaimed Antaibao opencast mine areas.Firstly,effects of different reclamation patterns on soil bacterial and fungal community structure were investigated using polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)and clone sequencing,respectively.Bacterial and fungal diversity index varied significantly with different reclamation patterns,respectively.The significantly highest and second highest bacterial diversity were found in Populus simonii and Pinus tabuliformis stand,respectively.And the significantly highest fungal diversity was found in Ulmus pumila stand.No significant change respectively of soil bacterial and fungal community structure was observed in the similar reclamation years based on cluster analysis and principal component analysis(PCA).Pearson correlation analysis demonstrated that bacterial and fungal diversity index did not significantly correlate with soil p H,organic carbon(OC),total nitrogen(TN),C/N as well as absolute water content(WC),respectively.Redundancy analysis(RDA)showed that soil pH,OC,TN,C/N as well as WC had no significant effect on soil bacterial and fungal community succession,respectively.Nitrospira,Sphingomonas,Arthrobacter,Streptomyces,Brachybacterium,Rhizobium,Devosia,Microvirga as well as Mesorhizobium which play important roles in the nitrogen cycle,symbiotic fixation with plants,degradation of polycyclic aromatic hydrocarbons and other organic matter,were identified by the clone sequencing of the bacterial DGGE bands.In addition,Phoma,Fusarium,Myrothecium,Pycnoporus as well as Cladosporium which play important roles in heavy metal adsorption,laccase production to degrade polycyclic aromatic hydrocarbons and other organic matter,symbiosis with plants,were identified by the clone sequencing of the fungal DGGE bands.There were also some pathogenic fungi,endangering the health of animals,plants and even human beings.Secondly,soil N-related functional microbial abundance(including AOB-amoA and AOA-amoA genes related nitrification,nitrogen-fixing microbial nifH gene and denitrifying bacterial nirS as well as nirK genes related denitrification)under different reclamation patterns were investigated using quantitative real-time polymerase chain reaction(qPCR).In addition,effects of different reclamation patterns on soil functional microbial community structure(including AOB-amoA as well as AOA-amoA genes related nitrification and nitrogen-fixing microbial nifH gene)were also investigated using PCR-DGGE.Abundance of AOB-amoA,AOA-amoA,nifH,nirS and nirK varied significantly with different reclamation patterns,respectively.The highest abundance of AOB-amoA,AOA-amoA,nifH,nirS and nirK was found in U.pumila(20 years),P.simonii(22 years),P.tabuliformis(5 years),Hippophae rhamnoides(8 years)and Armeniaca vulgaris(20 years)stand,respectively.The abundance of AOB in soil samples was lower than AOA,and the abundance of nirS was similar to nirK.No significant change of the relative abundance of each functional gene with the extension of reclamation years.The diversity of AOB and AOA varied with different reclamation patterns,but the difference was not significant,respectively.And nitrogen-fixing microbial diversity index varied significantly with different reclamation patterns.The highest diversity of AOB and AOA were both found in Picea asperata(20 years)stand,the significantly highest diversity of nitrogen-fixing microbes was found in P.tabuliformis(5 years)stand.Pearson correlation analysis demonstrated that the abundance of AOA-amoA,nifH and nirS were correlated with WC,and the former was extremely significantly negative correlation,the latter two were significantly positive correlation.The diversity index of AOB-amoA was only significantly negative correlated with TN.However,the diversity index of AOA-amoA did not just correlate with TN,and it was significantly positive correlated with soil p H,and it was significantly negative correlated with OC,C/N and WC,respectively.The diversity index of nitrogen-fixing microbial nifH was significantly negative correlated with C/N.RDA showed that soil pH,OC,TN,C/N and WC had different effects on the abundance and diversity of functional microbes,respectively,and soil OC and TN both affected them significantly(P<0.01),WC affected them significantly(P<0.05).Therefore,the results demonstrated that soil pH,WC,OC,TN and C/N may be important environmental factors affecting functional microbial community.Finally,the soil comprehensive fertility index(IFI)was calculated by PCA analysis of soil physiochemical factors,microbial diversity and abundance.The soil IFI was much higher than soil plots with shorter reclamation years.The soil IFI of Robinia pseudoacacia,P.tabuliformis,P.simonii and U.pumila were higher than others,which would be used as the optimum vegetation for improving the soil fertility in reclaimed mine areas.