Heritability for enteric methane emission from Danish Holstein cows using a non-invasive FTIR method

Abstract

Introduction.Methane is an important atmospheric greenhouse gas. Around 500-600 Tg of CH 4 /yr is emitted globally. Of this, about 74% is biologically generated almost exclusively by methanogens that belong to the phylum Euryarchaeota and grouped under six orders. Ruminant methanogens generate 13-19% of global methane output which is the largest of anthropogenic emissions. Among the rumen methanogens, Rumen Cluster 'C' (RCC), an unclassified and uncultured archaeal clade, represents 15.8-80.9% of rumen archaea (Janssen and Kirs, 2008), which has the potential to contribute significantly to ruminant derived methane. Phylogenetically, RCC distantly affiliate to a non-methanogenic order -Thermoplasmatales. Analysis of 16S rDNA identified members of RCC in diverse habitats including digestive tracts of insects, birds, cattle, pigs, camels, macropod marsupials and man. However, their position as methanogens was unresolved because of a lack of pure cultures, despite methane producing, non-axenic archaeal cultures being enriched from these sites. Here, we report the isolation and characterisation of the first methanogen from the RCC clade, strain DOK-1 in pure culture, that in turn could help understand the role of this large group in the rumen. We propose the nomenclature Methanoplasma gallocaecorum strain DOK-1, for this novel methanogen. Material and methodsCaecal contents from broiler chickens were anaerobically pooled and inoculated into 10 ml Balch tubes containing anaerobic BRN enrichment media (Rea et al., 2007). Methylotrophic methanogens were enriched by adding methanol, mono-, di-and trimethylamine HCl. Cultures were grown at 39 o C under 150 kPa of 80:20 H 2 :CO 2 and monitored for CH 4 production by GC and sub-cultured every 3-5 days for several transfers. Cultures of a 10-fold serial dilution with CH 4 production at the highest dilution were further purified by antibiotics. Methanogens were also monitored by phase-contrast microscopy and a qPCR using mcrA and total bacterial primers. Finally, a persistent bi-culture consisting of a methanogen and a bacterium was obtained. Isolation was achieved by inoculation of serial dilutions of cultures into agarose containing solid media. Several, single colonies were picked and cultured in liquid media to determine purity. Genomic DNA from these cultures were amplified using archaeal 16S primers 86F-1340R; direct sequencing of PCR products confirmed sequence alignment with Thermoplasmatales affiliated novel methanogens. Nutritional and physiological requirements and morphologic features of the isolate were studied; cells in late exponential phase were freeze-dried, fixed and analysed by transmission electron microscopy. Results and ConclusionCells did not fluoresce under UV and appeared round (cocci) by both phase contrast and TEM microscopy measuring ~650-1200 nm in diameter. They are non-motile and non-flagellated with a fragile proteinaceous cell wall with no detectable S-layer. EM showed a delicate cell wall, a simple plasma membrane and a nondescript cytoplasm o...