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• Methane emissions from ruminant livestock are proposed to contribute ~10 to 20% of annual global greenhouse gas emissions. • Recent efforts have shown that "low methane emitting" animals can be identified both within and across species, including nondomesticated herbivores. • Methanogens are also present within the human gut and some other body sites, and variations in their diversity and/or abundance has been linked to some functional gastrointestinal disorders and non-communicable chronic diseases. • Recent advances in "-omics" technologies have provided new insights of the roles that methanogens play in both feed digestion and host-microbe interactions. • Although much research and understanding has been derived from the study of the numerically dominant Methanobrevibacter genus, the recent studies outlined above have shown that other, "heterotrophic" methanogens warrant greater attention. • Here, we provide a brief overview of our recent research of the Methanosphaera genus and outline the possible consequences associated with its presence in the gut of livestock, native Australian herbivores, and humans.

Quantitative real-time PCR (qPCR) has become a popular method for estimation of meth- anogen abundance in the ruminant digestive tract. However, there is no established method in terms of primer choice and quantification, which means that results are variable and not directly comparable between studies. Archaeol has been proposed as an alternative marker for methanogen abundance, as it is ubiquitous in methanogenic Archaea, and can be quantified by gas chromatography-mass spectrometry (GC-MS). The aim of this experiment was to compare total metha- nogen populations estimated using the new archaeol approach with estimates based on qPCR. Specific prim- er sets and probes were used to detect dominant ruminal methanogen species Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanosphaera stadt- manae, and total methanogen populations. There was variation in the relationships among total methanogen abundance estimates based on archaeol and qPCR. In addition, the universal methanogen primers appeared to preferentially amplify genes from M. smithii. Archaeol had the strongest relationship with the dominant rumen methanogen M. ruminantium, whereas the total metha- nogen primers had a comparatively weak relationship with archaeol. Archaeol analysis was a useful adjunct to molecular biology methods, but it seems that a valid specific primer for M. ruminantium would be more use- ful than a biased primer for total methanogens.

Aims To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. Methods and Results DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Conclusions Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Significance and Impact of the Study Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 °C) and all other methanongens (84-86 °C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.