Comparative genomics of rumen methanogens : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

Methane (CH4) emissions from agriculture represent around 9% of global anthropogenic greenhouse gas emissions. The single largest source of this CH4 is animal enteric fermentation, predominantly from ruminant livestock, where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce CH4 emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop CH4 mitigation technologies. To gain insights into the role of methanogens in the rumen environment, two methanogens have been isolated from ovine rumen and their genomes were sequenced: methanogenic archaeon ISO4-H5 of the order Methanomassiliicoccales and Methanobrevibacter sp. D5 of Methanobrevibacter gottschalkii clade. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M (CoM), and requires external supply of CoM to survive. Methanobrevibacter sp. D5 is a hydrogenotrophic methanogen predicted to utilise CO2 + H2 and formate as substrates. Comparisons between the available Methanobrevibacter genomes has revealed a high conservation in energy metabolism and characteristics specific to each clade. The coexistence of different Methanobrevibacter species in the rumen may be partly due