The temperature program for the archaea consisted of denaturation at 95°C for 2 min, followed by 40 cycles consisting of 95°C for 15 s, annealing and extension at 60°C for 1 m. Melting curve analysis
was conducted over a range of 60 to 95°C to assess specificity of the amplification products. The 10-fold dilution series of the standard plasmid for the respective target was run along with the samples. Amplification SN-38 of each sample was performed in triplicate. Quantification was based on standard curves obtained from the amplification profile of known concentrations of the standard plasmid for the respective target. The total Akt targets numbers of methanogens per gram wet weight or ml or cm2 were determined using ABI SDS software (Applied Biosystems, Foster City, CA, USA) and according to dilution factor and volume of DNA extracts. Methane detection Methane was detected by GC (Shimadzu, gas chromatograph GC-14 B, Japan) according to the method described in our previous study : capillary column (Supelco, Column No. 41491-03B, US) temperature 80°C, vaporizer temperature 100°C, flame ionization detector temperature 120°C, carrier gas (N2) pressure 0.05 MPa, H2pressure 0.05 MPa and air pressure 0.05 MPa. Identification of anaerobic fungus The anaerobic fungi in the cultures were examined by microscopy (Eclipse 80i, Nikon, Japan) with DAPI (4’, 6 diamidino-2-phylindole) staining according to our previous study
. An aliquot of 1 ml 3-day old culture was treated with Etomidate 1 μl, 500 μg/ml DAPI (Sangon Biotech (Shanghai) co., Ltd., Shanghai, China), stored in dark room for 5 min, and then examined by fluorescence microscopy. Statistical analysis selleck compound All data were analyzed by Tukey’s analysis of one-way ANOVA of SPSS 18.0 (SPSS, Chicago, IL, USA) at a 95% significance level. Acknowledgements This work was supported by grants from the Natural Science
Foundation of China (31072052, 31101735), China-Australia Project (2010DFA31040) and the Fundamental Research Funds for the Central Universities (KJ2013018, KYZ201312). References 1. Tajima K, Nagamine T, Matsui H, Nakamura M, Rustam I, Aminov RI: Phylogenetic analysis of archaeal 16SrRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol Lett 2001, 200:67–72.PubMedCrossRef 2. Wright ADG, Williams AJ, Winder B, Christophersen C, Rodgers S, Smith K: Molecular diversity of rumen methanogens from sheep in Western Australia. Appl Environ Microbiol 2004, 70:1263–1270.PubMedCentralPubMedCrossRef 3. Wright ADG, Toovey AF, Pimm CL: Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea. Anaerobe 2006, 12:134–139.PubMedCrossRef 4. Wright ADG, Auckland CH, Lynn DH: Molecular diversity of methanogens in feedlot cattle from Ontario and Prince Edward island Canada. Appl Environ Microbiol 2007, 73:4206–4210.PubMedCentralPubMedCrossRef 5.