Study On The Response Feature Of Rhizosphere Microbial Community In The Process Of The Pongamia Pinnat-Rhizobium For In Situ Remedating V-Ti Magnet Tailings

To evaluate the in-situ remediation efficiency of V-Ti magnetite tailings by the Pongamia pinnata-rhizobia symbiosis system and the dynamic change of microbial community structure in rhizosphere soil during the remediation process,mine tailings grown with Pongamia pinnata plants for 0,1 and 2 years were taken as the objects for the investigation of growth status,nitrogen content,and the concentration and translocation of heavy metals;Using high-throughput sequencing technologies for the V4 area of 16S r RNA genes in the rhizosphere soil,the microbial community structure of the Pongamia pinnata-rhizobia symbiosis system in V-Ti magnetite tailings during the in-situ remediation was revealed.In combination with physical and chemical properties and heavy metal content in rhizosphere soil,the correlation between the microbial community structure in rhizosphere soil and environmental factors was analyzed;the gradient denatured gel electrophoresis(DGGE)and fluorescent quantitative PCR technology were applied to study the diversity of Pongamia pinnata rhizosphere-associated nitrogen-fixing microbes and to reveal the effects on nitrogen fixation microorganisms in rhizosphere soil by Pongamia pinnata-rhizobia symbiosis system.The results are as follows:During the in-situ remediation progress using the Pongamia pinnata-rhizobia symbiosis system in V-Ti magnetite tailings,the available,total,and microbial biomass nitrogen were lifted,while microbial biomass carbon,phosphatase,catalase and sucrase were increased after a decline;the nitrogen increase,total phosphorus,total potassium and organic matter were reduced by the year,while the effective phosphorus and urease were increased;The effective Fe in rhizosphere soil showed a significant decrease year by year,and the effective Fe after 1 year and 2 years remediation were reduced respectively by41.13%and 16.90%compared with the soil without remediation.During the in-situ remediation of the tailings soil,Pongamia pinnata was well developed,leading to a yearly increase in plant height,root length,biomass,superoxide dismutase(SOD)and nitrogen content.After 2 years remediation,the concentration of heavy metal Cu was 23.85 mg/kg,and the translocation of heavy metal Ni was 1120.28mg/kg.This indicates that Pongamia pinnata,which has strong translocation ability for Ni,can be applied to the remediation of Ni-contaminated soil.During the in-situ remediation progress of the Pongamia pinnata-rhizobia symbiosis system in V-Ti magnetite tailings,the total DNA of rhizosphere soil was extracted and the high-throughput sequencing of 16S r RNAV4 area was used to analyze the relative abundance of species,which showed that Proteobacteria and Firmicutes increased after a decrease at the beginning,while Actinobactetia and Acidobacteria decreased after an increase;Acinetobacter,Sphingomonas and Massilia were the dominant bacterial species,while Rhizobium increased continuously.The analysis of Alpha diversity manifested that after 1 year of remediation,the richness and uniformity of the rhizosphere microbial species decreased.After 2 years of remediation,the maximum species richness and uniformity were reached.Beta diversity analysis incidated that 1 year later,the change of Pongamia pinnata rhizosphere microbial community structure was higher,and the analysis of CCA and RDA at the genus level combining with environmental factors showed the change of the bacterial community structure has significant correlation with catalase,the effective Fe,and microbial biomass nitrogen(P<0.05).The diversity of microbial nitrogen fixers in Pongamia pinnata rhizosphere soil was varied,as revealed by the DGGE and gene sequencing technologies,which identified different genera including Bradyrhizobium sp.,Rhizobium sp.,Azotobacter sp.and Azohydromonas sp..It was found that two years remediation caused the nitrogen fixing bacteria in the symbiotic rhizosphere soil to be the most abundant.Fluorescence quantitative q PCR technique was employed to perform relative quantification of nif H gene in different remediation years,which showed the copy number of nif H gene first decreased and then increased in the range of 4.92×10⁷-3.81×10⁸ copies·g^-1,and that two years later the nif H gene copy number achieved the highest,in consistent with the change of the rhizobial community.This explained that nitrogen-fixing microbial community structure had a dynamic change during the remediation progress.