Bacterial Diversity In Rhizospheric Soils Of Predominant Plants Growing In Copper Mining Wasteland And The Effects Of Copper-resistant Bacteria On The Accumulation Of Copper From Soil By Maize

Phytoremediation is an emerging as a cost-effective and environment-friendly technology for remediation of heavymetal-contaminated soils. However, phytoremediators, the plants used for remediation, often have a slow growthrate and produce limited amounts of biomass.Using chemicals to enhance phytoremediation is comparatively available but usually expensive and unfriendly to environment. Therefore, it is very important to search for a microbe-strengthened phytoremediation approach to enhance plant remediation economically and effectively.In this study, bacterial diversity of rhizospheric soils of predominant plants growing in copper mining wasteland was analyzed at first through Denaturing gradient gel electrophoresis (DGGE). The results showed great diversity of heavy metal resistant bacteria in rhizospheric soils while no apparent microbe community structure differences were observed among different rhizospheric soils, with a similarity of more than 87%. Combining with traditional microbe isolating method of streak plating, 89 copper-resistant bacterial strains (Cu 50 mg·L^-1) were screened out from 11 rhizospheric soil samples. Further more, 17 strains with higher copper resistance (Cu 200 mg·L^-1) and copper-solubilizing ability were screened out by increasing Cu concentration gradually and Cu solubilization experiment. The analysis of 16S rDNA sequences of these 17 bacterial strains revealed abundant genetic diversities; copper solubilization experiments in solution culture suggested their functional diversities; the intrinsic ability for the production of ACC deaminase, IAA, siderophore, and so on, suggested the metabolic diversities among them. The results of the influence of temperature, pH, other heavy metals, antibiotics on the growth of the 17 strains also showed abundant diversities.Four strains JYC17, MT16, YAH27 and HQN2 were selected from the 17 strains with higher biomass, more acid production, more effectively copper solubilization, stronger survival ability and growth-promoting ability for further studies. Copper solubilization of the four strains was investigated in solution culture containing Cu₂(OH)₂CO₃ and Cu(OH)₂. The results showed that they could not only endure copper but also solubilize hardly-soluble copper. The pH value of liquid YN medium added with Cu₂(OH)₂CO₃ and Cu(OH)₂ in the bacterial inoculation treatments was decreased by 2.29 - 3.04 unit and 0.19 - 2.64 unit respectively, at the same time the concentration of available copper was increased by 136% - 402% and 103% - 218% respectively. Deeper studies showed that the organic acid produced by the four strains were different and copper could promote the production of organic acid. All the four strains could significantly increase the concentration of available copper in the soil (added Cu₂(OH)₂CO₃ or added Cu(OH)₂ or copper-contaminanted soil) compared to the control, HQN2 was the best and YAH27 was the second. After 10 days' cultivation, the amount of inoculated bacteria reached 10⁶ - 10⁷ cfu·g^-1 soil. As for the strong buffer capacity of the soils, the pH values of the inoculated soils only decreased a little compared to the control.JYC17-gfp, MT16-gfp, YAH27-gfp and HQN2-gfp with gfp marker gene were gained by tri-parental mating. Studies showed that the gfp gene could steadily exist in the labeled strains and did not affect their copper-solubilizing ability. Colonization experiment showed the labeled strains JYC17-gfp, MT16-gfp, YAH27-gfp and HQN2-gfp could not colonize in the inner root of maize, but could colonize on the surface of the root and could promote the growth of maize at seeding stage.The pot experiments demonstrated that the four strains JYC17-gfp, MT16-gfp, YAH27-gfp and HQN2-gfp could promote the growth of maize. Especially, strain JYC17-gfp had the best effects which could apparently increase the biomass of maize shoots by 23.0% and significantly increase the biomass of maize roots by 14.7%. The content of available Cu in the inoculated maize rhizospheric soil increased apparently and the Cu accumulated by the plants increased at the same time. Compared to the control, the concentration of copper in the maize shoots increased by 26.7% - 63.3%, and increased by 25.6% - 45.2% in the maize roots. The accumulated Cu by maize always aggregated in the root and was hard to transmit to the above-ground parts, the inoculation had a positive effect for Cu²⁺ transmission from root to shoots.