## 2 Materials and Methods
### Experimental Design
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Treatment** & **Amendments** & **Amendments** \\ \hline CK & control1 & mine soil \\  & control2 & mine soil + sawdust * \\ \hline \multirow{6}{*}{L1} & lime1 & mine soil + limel (25 t ha\({}^{-1}\)) \\  & lime1+NPK & mine soil + limel (25 t ha\({}^{-1}\)) + NPK compound fertilizer (150 kg ha\({}^{-1}\)) \\  & lime1+P & mine soil + limel (25 t ha\({}^{-1}\)) + phosphate fertilizer (300 kg ha\({}^{-1}\)) \\  & lime1+Rs & mine soil + limel (25 t ha\({}^{-1}\)) + River sediment (30\%, w:w) \\  & lime1+NPK+Rs & mine soil + limel (25 t ha\({}^{-1}\))+ NPK compound fertilizer (150 kg ha\({}^{-1}\)) + River sediment (30\%, w:w) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Experimental design for pot experiment. DNA Extraction, 16S rRNA Polymerase Chain Reaction (PCR) and Bacterial Community T-Restriction Fragment Length Polymorphism (RFLP) Profiles Analysis  
Genomic DNA was extracted using a modification of the protocol described by [PERSON] et al. [19]. In brief, 5 g soil was added to 10 mL of buffer [100 mM Tris-HCl (pH 8.0), 100 mM ethylene diamine tetraacetic acid (EDTA) (pH 8.3), 100 mM phosphate buffer (pH 8.0), 1.5 M NaCl, 1% cetyltrimethylammonium bromide], 50 uL of proteinase K (20 mg mL\({}^{-1}\)) and 60 uL of lysozyme (100 mg mL\({}^{-1}\)). Samples were incubated at 37 \({}^{\circ}\)C with shaking at 250 rpm for 30 min. 1.5 mL of 20% sodium dodecyl sulphate was added, and the tubes were gently agitated and then incubated at 65 \({}^{\circ}\)C for 2 h. Samples were centrifuged at 10000 xg for 10 min. The supernatant was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). DNA was precipitated with 0.6 volumes of isopropanol, washed with 1 mL of 70% ethanol and resuspended in sterile, deionized water. The quantity of the DNA extracted was checked with a spectrophotometer (NanoDrop). 16S rRNA gene fragments were amplified from the bulk DNA sample using the universal primers 533F (5'-GTGCCAGCMGCCGGTAA-3') and 1492R (5'-GGTTACCTTGTTACGACT T-3') [20]. PCR reactions were performed in 40 uL aliquots containing 20 uL of Mix (0.1 U TaqMan DNA polymerase uL\({}^{-1}\), 500 uM dNTP each, 20 mM Tris-HCl (pH 8.0), 100 Mm KCl, 3 mM MgCl\({}_{2}\), obtained from Beijing Tiangen Bio-Engineering and Technologies, China), 0.4 uL of each primer, 18.2 uL of dH\({}_{2}\)O and 10 ng extracted total soil DNA. The thermocycling conditions were as follows: a hot start at 94 \({}^{\circ}\)C for 4 min (1 cycle), 94 \({}^{\circ}\)C for 1 min, 55 \({}^{\circ}\)C for 1 min, 72 \({}^{\circ}\)C for 2 min (35 cycles), and a final extension at 72 \({}^{\circ}\)C for 10 min. PCR products were first visualized on a 1% agarose gel and purified using a High Pure PCR Product Clean Up Kit (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer's instructions. The purified PCR products were digested enzymatically as follows: approximately 10 uL of PCR product was added to a reaction mixture containing sterile Millipore water, 30 U of restriction endonuclease Msp I / Rsa I and 2 uL of corresponding enzyme buffer. Digests were performed in a final volume of 20 uL, incubated in a water bath at 37 \({}^{\circ}\)C for 4-6 h and denatured 65 \({}^{\circ}\)C for 20 min. Terminal restriction fragments (T-RFs) were determined by electrophoresis using a ABI3730 automated sequencer (USA), and analysis of fragment profiles was performed using Peak-Scanner Software v.1.0. Only fragments with a fluorescence \(>\) 1% of the total fluorescence were included in the analyses. Bacterial community structure was estimated using the Shannon-Wiener diversity index (H) which was calculated as described by [PERSON] et al. [21] using the following formula:  
\[H=-\sum_{i=1}^{n}\left(P_{i}\ln P_{i}\right)\]  
where p\({}_{i}\) is the relative abundance of each T-RF in the total sum and n is the number of detected T-RFs.