Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile by fluorescence in situ hybridization

The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F-420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium mobile, Methanobrevibacter ruminantium and Methanobrevibacter smithii. Epifluorescence microscopy (F-450 autofluorescence) and irt situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.