2.4. DGGE and phylogeny
The 233 bp-long pufM fragments from the water and sediment samples were separated in a 45-70% and 40-60% (w/v) denaturing gradient, respectively into a 0.8% (w/v) polyacrylamide gel. The electrophoresis was performed at 80 V, and 60 oC for 16 h. After electrophoresis, the polyacrylamide gel was stained using the SYBR Gold nucleic acid stain for 20 min. The images were detected and captured on a UV transilluminator. The pufM fragments were cut from the gel using Gel Cutting Tips (Cleaver Scientific, England) and re-amplified with the pufM forward and pufM750-AT-M13 primers. The PCR products were purified using the Gel/PCR DNA fragment extraction kit (Geneaid, Taiwan) according to the manufacturer's instructions and then sequenced with pufM750-AT-M13 as sequencing primers using the 1st BASE Laboratories Sdn Bhd (Malaysia) and were compared with the GenBank database in the NCBI website. Phylogenetic analysis was con structed by neighbor-joining using the free software MEGA 5.