title: Bacterial community compositions in the horizontal subsurface flow constructed wetland without pre-aeration
design: A comprehensive survey on the bacterial community compositions inside the horizontal subsurface flow constructed wetland withot pre-aeration was made by pyrosequencing. For each sample, we amplified V1–V3 region of bacterial 16S rRNA genes using a broadly conserved primer set (27F and 533R). PCR reactions were carried out in triplicate 20-μL reactions with 0.4 μM forward and reverse primers, 1-μL template DNA, 250 nM dNTP and 1× FastPfu Buffer. All dilutions were carried out using certified DNA-free PCR water. Thermal cycling consisted of initial denaturation at 95°C for 2 min followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension of 5 min at 72°C. Replicate amplicons were pooled and visualized on 2.0 % agarose gels using SYBR Safe DNA gel stain in 0.5× TBE. Amplicons were purified using AxyPrep™ DNA Gel Extraction Kit (Axygen) according to the manufacturer’s instructions. Then the DNA was quantified using QuantiFluor™-ST (Promega Corporation, CA, USA). Pyrosequencing was carried out on a 454 Life Sciences Genome Sequencer FLX Titanium instrument (Roche).