| Soil is an important container of heavy metals accumulation, heavy metals in which can be enrichmented for times by plants, animals through the food chain. The toxicity of heavy metals in soil is not only related to the total concentration, but to a greater extent is determined by its speciation distribution. Microbes is the most active part of organic components, and ecological system in soil, which plays an important role in the promotion of soil quality and plant health, and is considered as the most sensitive biological indicators of soil quality. The ecological effects of heavy metals on soil organisms, on one hand had bad performance in the bio mass, metabolic activity, reducing the number and diversity of species; On the other hand, a large number of tolerance taxa could grow and resistant to heavy metal contamination, because of the choice stress of heavy metals and biological adaptive responses. Change of microbial community composition and diversity may reflect soil variability and soil quality, based on the quantity and function of microbes. In order to better understand the health status of the soil, the impact of heavy metal pollution on microbial community structure, it is very necessary to study the microbial diversity, microbial community composition and their interaction with heavy metals in the contaminated soil further. In this study, we chose the 4# tailing of Dexing Copper Mine as object,16 samples were obtained, including tailing samples, vegetable field soil samples and grain field soil samples, and were investigated.(1) The distribution and chemical speciation of heavy metals in mine tailing and near soils were investigated. The total content of heavy metals and speciation of heavy metals (Cu, Cd and Zn) were investigated by atomic emission or atomic absorption spectroscopy. Tessier's extraction scheme was used in the investigation of the mobility and transport of the metals. The potential risk of environmental pollution of heavy metals Cu, Cd and Zn was assessed, both based on total concentrations, and on bioavailable fractions according to a Risk Assessment Code (RAC) for the first time, in these long-term heavy metal-polluted tailing soil samples. High levels of heavy metals were detected in samples, showing a certain extent of dispersion of heavy metal pollution from the mine tailing. The Tessier's sequential extraction results showed that Cu was mainly associated with the fraction bound to organic matter (ORG) and residual fraction (RES). Cd was mainly associated with the exchangeable fraction (EXC) and Zn appeared mainly associated with the residual fraction (RES) in the samples. According to the Risk Assessment Code (RAC), cadmium showed high to very high environmental risk, which agreed with the heavy pollution classification (III) proposed by the standard (GB 15618-1995), China, in almost all the samples; While copper and zinc showed low to medium risk in many samples, which disagreed with the classifications proposed by the standard GB 15618-1995 for total metal concentrations. The results revealed that cadmium pollution is serious in the studied area and has high environmental risk, should be paid more attention as soon as possible. Besides, it may suggest that bioavailable metal fractions should be included in an adequate criterion for environmental risk assessment, not only based on the total metal contents.(2) Microbial diversity in samples. Firstly, the number of culturable heterotrophic bacteria in 16 samples was investigated by plate counting. It was found that, in the tailing samples T4 and T7, the number of culturable heterotrophic bacteria was the least, about 0.5 X 107cfu/g dry soil; in the vegetable field soils it ranged from 1.40 to 2.75 X 107cfu/g dry soil, and in the grain field soils it ranged from 0.60 to 3.60 X 107 cfu/g dry soil, were higher than that in tailing samples.16S rDNA V3 variable segments were obtained and separated by DGGE (denaturing gradient gel electrophoresis). UPGMA cluster analysis of the DNA fingerprint was carried out by Quantity One software, and the result showed that, samples T4 and T7 has similarity 56.4%; vegetable field soil samples V11, V13, V15, V18 and V20 were gathered, and were separated form the sample V8, which indicated the big difference of microbial diversity between sample V8 and other samples; in grain field soil samples, samples G10, G12, G14 and G16 were together, and were separated from clustered samples G17,G19 and G21.Diversity index of the samples (Shannon-Weaver index H) was calculated, it found that the greatest diversity in the samples that were at middle distance away from the mine tailing, such as vegetable field soil samples V13, V15, grain field soil samples G12 and G14. In these samples, the contents of heavy metals copper and zinc are not high (not exceed the national soil quality standard II), but also are not the lowest. On contrary, in samples which has the highest contents of heavy metals copper and zinc (such as vegetable soil sample V8 and grain soil sample G10), as well as the samples which has the lowest contents of heavy metals copper and zinc, and were 10 km distance away from the tailing (such as vegetable soil sample V20 and grain soil sample G19), the microbial diversity index H was similar to each and were lower than the maximum. The results may indicate that the influence of heavy metals on microbial diversity is not a simple linear relationship with heavy metals concentration, and in a certain concentration range, heavy metals may contribute to the development of microbial diversity.(3) Composition of microbial communities in samples. The compositions and structures of microbial communities in the soil samples were determined by a PCR-based cloning approach (restriction fragment length polymorphism, RFLP). A total of 236 OTUs (operational taxonomic units) were obtained in these samples. After sequencing, the bacterial species in each sample were determined as:T4 (9 species), V8 (56 species), V20 (40 species), G10 (56 species) and G19 (46 species).The main dominant bacterial species in these samples were:uncultured Pseudoxanthomonas sp. clone GI5-005-C10, Burkholderia sp.383, uncultured Acinetobacter sp. clone TCCC 11180 and so on, more than ten kinds. The results of phylogenetic analysis revealed that a total of 207 bacterial species in the five samples fell into fifteen putative phylogenetic divisions, which were Acidobacteria, Actinobacteria, Bacteroidetes and so on. The distribution of dominant groups in samples was different. In sample T4, the predominant group wasγ-proteobacteria, representing 86% of the total clones in T4 bacterial library; in sample V8, the dominant groups wereγ-proteobacteria, a-proteobacteria,δ-proteobacteria, Planctomycetes, Acidobacteria and Bacteroidetes, representing 14.5%,12%,8%, 14.5%,10% and 9.7% of the total clones in V8 bacterial library, respectively; in sample V20, groups y-proteobacteria (25%),β-proteobacteria (16%), Cyanobacteria (12%),α-proteobacteria (10%) and Acidobacteria (10%) were dominant bacterial phylogenetic divisions; in sample G10, the dominant groups wereβ-proteobacteria (22.5%),γ-proteobacteria (10%),α-proteobacteria (10%), Chloroflexi (10%) and Firmicutes (10%); in sample G19, the dominant groups were Acidobacteria,β-proteobacteria,α-proteobacteria, Chloroflexi and Planctomycetes, representing 22%,17%,14%,13% and 11% of the total clones in G19 bacterial library, respectively. Overall, the differences of bacterial community composition in five samples were apparent. T4 tailing sample had the least bacterial diversity, the community structure in it was relatively simple, the dominant group was single and predominance was obvious; in vegetable and grain field soil samples, a marked increase in bacterial diversity was investigated, the number of communities increased, the structure of microbial community composition was more complex, dominant groups were not obvious. Compared with the standard of microbial diversity and the average community composition in typical healthy soil samples proposed by Janssen, the studied soil samples with long term heavy metal pollution, in which the composition of microbial community structure has occured an obvious change, indicating that the soils may be in an unhealthy state.In addition, the correlation between total concentration and bioavailability of heavy metals Cu, Cd, Zn and microbial groups for the impact have firstly been investigated by PC A in this study. We found that different heavy metals and different forms of heavy metals had different effects on the distribution of microbial communities. Focus on considering the relationship between bioavailability of heavy metals content (ie, exchangeable and carbonate-bound heavy metals) and microbial communities, found that exchangeable heavy metals copper and zinc was correlated to bacterial populations Act (Actinobacteria), Bet(β-proteobacteria), Chl (Chlorobi), Chlo (Chloroflexi) and Fir (Firmicutes); Carbonate-bound copper and zinc was closely related to bacterial groups Bac (Bacteroidetes) and Ver (Verrucomicrobia); correlation between exchangeable, carbonate-bound heavy metal cadmium and bacterial population Gam (γ-proteobacteria) is particularly significant. From the results, we believed that according to the correlation between the microbial communities and heavy metal bioavailability, the changes of microbial community structure might be used to indicate the bioavailability of heavy metals, such as changes in the number of Gam (y-proteobacteria) may indicate the changes of cadmium bioavailability contents.(4) Isolation and characterization of highly heavy metal resistant bacterial strains. Highly heavy metal resistant indigenous bacterial strains DX-T3-01, DX-T3-02 and DX-T3-03 were isolated from the tailing sample by plating method. The strain DX-T3-01 exhibited high tolerance to cadmium:grow well on YTPG agar plates with 10 mM Cd2+and in liquid medium with 18 mM Cd2+; The strain DX-T3-02 exhibited high tolerance to copper:grow well on YTPG agar plates with 3 mM Cu2+ and in liquid medium with 6 mM Cu2+; The strain DX-T3-03 was highly resistant to zinc and could endure 35 mM Zn2+ on YTPG agar plates and 30 mM Zn2+ in liquid medium. On the basis of 16S rDNA sequencing, BLAST and phylogenetic analysis, the strains were identified as Ralstonia pickettii strain DX-T3-01, Methylobacterium sp. strain DX-T3-02 and Sphingomonas sp. strain DX-T3-03, respectively. This study supplied potential indigenous bacterial materials for tailing bioremediation studies in the future. Compared to other reported strains, the bacteria isolated in this study had very significant advantages in heavy metal resistance, and was expected to be developed into excellent bacterial materials in bioremediation of heavy metals.The DGGE method was also applied to detect the distribution of these three bacterial strains in the samples. It was found that in different samples the distribution and abundance of different bacterial strains was different obviously. Generally, the characteristic band of Ralstonia pickettii strain DX-T3-01 was detected in tailing samples and several samples near the tailing, with high brightness, which indicated the big amount of Ralstonia pickettii strain DX-T3-01 and it might be one of the dominant bacterial species in these samples; The characteristic band of Methylobacterium sp. strain DX-T3-02 was only detected in samples T4 and V8 with great brightness, and was detected faintness or not detected at all in other samples, which might indicate the strain could be strict to the characteristics of samples, and the speciality of this strain was obvious. While the characteristic band of Sphingomonas sp. strain DX-T3-03 was detected in all of the samples with low brightness, which might indicate that this bacterial strain was spread widely in environmental samples and not the predominant bacterial species in these samples. This method may be utilized measuring the heavy metal pollution, seeking for heavy metal microbial marker, and developing new fast detecting technology in future.
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