Fragments of the methanogen DNA (~470 bp) were amplified by PCR using mcrA gene specific primers. Primer sequences for mcrA gene are as follows, mcrA F 5′- GGTGGTGTACGGATTCACACAAGTACTGCATACAGC-3′,mcrA R 5′-

TTCATTGCAGTAGTTATGGAGTAGTT-3′. PCR amplification was carried out in 50 pl reaction mixture contained within 0.2 mL thin walled micro-tubes. Amplification was performed in a TC-XP thermal cycler (Bioer Technology, Hangzhou, China). The reaction mixture contained 5 pL of 10 x PCR amplification buffer, 200 pM of each dNTP, 0.8 pM of each primer, 2 pL of template DNA and 2.5 U of FastStart Taq DNA polymerase (Polymerase dNTPack; Roche, Mannheim, Germany). The initial enzyme activation and DNA denaturation were performed for 6 min at 95°C, followed by 5 cycles of 30s at 95°C, 30s at 55°C and 30s at 72°C, and the temperature ramp rate between the annealing and extension segment was set to 0.1°C/s because of the degeneracy of the primers. After this, the ramp rate was set to 1°C/s, and 30 cycles were performed with the following conditions: 30 s at 95°C, 30 s at 55°C, 30s at 72°C and a final extension at 72°C for 8 min. PCR products were visualised by electrophoresis in ethidium bromide stained, 1.5% (w/v) agarose gel.

Purified PCR amplicons (PCR purification kit; Qiagen, Venlo, Netherlands) were ligated into TOPO TA cloning vectors and transformed into chemically competent Escherichia coli TOP10F’ cells according to the manufacturer’s instructions (Invitrogen, Carlsbad, USA). Positive colonies were screened by PCR amplification with the primer set and PCR conditions described above. Plasmids were extracted using UltraClean 6 Minute Plasmid Prep Kit (MoBio, Carlsbad, USA), and nucleotide sequences of cloned genes were determined by sequencing with M13 primers in Macrogen company (Seoul, Korea). Raw sequences obtained after sequencing were BLAST analysed to search for the sequence identity between other methanogen sequences available in the GenBank database. Then these sequences were aligned by using CLUSTAL W in order to remove any similar sequences. The most appropriate substitution model for maximum likelihood analysis was identified by Bayesian Information Criterion implemented in MEGA 5.05 software. The

phylogenetic tree was constructed by the maximum likelihood method (Kimura 2- parameter model). The tree topology was statistically evaluated by 1000 bootstrap replicates (maximum likelihood) and 2000 bootstrap replicates (neighbour joining).

3. Results