design: The V3V4 region of 16S rDNA gene were amplified by PCR (94 C for 2 min, followed by 30 cycles at 98 C for 10 s, 62 C for 30 s, and 68 C for 30 s and a final extension at 68 C for 5 min) using the forward primer 341F (5 CCTACGGGNGGCWGCAG 3) and the reverse primer 806R (5 GGACTACHVGGGTATCTAAT 3). PCR reactions were performed in triplicate 50 L mixture containing 5 L of 10  KOD Buffer, 5 L of 2 mM dNTPs, 3 L of 25 mM MgSO4, 1 L of KOD Polymerase (Toyobo, Osaka, Japan), 1.5 L of each primer (10 M), and 100 ng of template DNA.