*3.4. High-Throughput Sequencing*

Wetland media (5 g) were collected at the bottom layer to analyze microbial community. The V4 region of the bacteria 16S rRNA gene was amplified by PCR (5-GTGCCAGCMGCCGCGGTAA-3 , 5-GGACTACHVGGGTWTCTAAT-3) according to previous reports [51,52]. Each PCR reaction was performed with 50 μL mixture containing 35 ng template DNA, 4 μL PCR Primer Cocktail (5 μM, Qiagen, Valencia, CA, USA), 25 μL PCR Master Mix (Qiagen), and ddH2O [53]. Illumina MiSeq platform was employed to purify and pool amplicons in equimolar amounts and paired-end sequenced (2 × 250) [51,54]. Data were analyzed with Microsoft Excel 2010, and statistical analyses were conducted by SPSS ver. 19.0.
