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    Identification of rumen methanogens, characterization of substrate requirements and measurement of hydrogen thresholds : a thesis presented in partial fulfilment of the requirements for the degree of Master 's in Microbiology
    (Massey University, 2012) Kim, Caroline Chae-hyun
    In New Zealand, exported farmed commodities derived from ruminants make up about one-third of the nation’s economy. However, farming ruminants creates a significant environmental impact by emitting methane which is a by-product of the microbial fermentation occurring in the rumen. Accumulated methane in the atmosphere is considered to be an important contributing factor to global warming and climate change. Methanogenic archaea, collectively called methanogens, inhabiting the rumen are responsible for the production of ruminal methane. These organisms are capable of anaerobically reducing CO2 to CH4, using H2, formate, methanol, a range of methyl-compounds, or acetate as electron-donors. Currently, all known methanogens that have been isolated from a diverse range of habitats are classified into 28 genera and 113 species based on the study of pure cultures and analysis of small subunit rRNA gene sequence data. Less than 10% of these species were isolated from the rumen and these reflect only a small portion of the true rumen methanogen diversity that has been determined by cultivation-independent methods. This project has been derived from the necessity to characterise genome sequences of a greater diversity of rumen methanogens than is currently covered in public culture collections. 14 methanogen strains were isolated as pure cultures and identified based on 16S rRNA and mcrA gene sequences in order to create a comprehensive phylogenetic tree comparing the genetic distances between the newly identified strains and the few named species. Strains 229/11, AbM4, M1, SM9, G16, D5, BRM9, YCM1, ISO3-F5, and A4 were then selected to be characterised for their substrate requirements for growth, by systematically omitting single or multiple components from the growth medium. Finally, the threshold levels of hydrogen, below which the methanogens fail to use it as a substrate, were measured for these strains by gas chromatography. Overall, the H2 thresholds of rumen methanogens fell within the range between 0.5 and 5.8 Pa. Methanobrevibacter, the most predominant group of methanogens occurring in the rumen, had relatively higher H2 thresholds compared to the genus Methanosphaera, a group of methanogens frequently isolated from New Zealand ruminants, and the genus Methanobacterium.
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    Investigation of rumen methanogens in New Zealand livestock : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand.
    (Massey University, 2010) Jeyamalar, Jeyanathan
    Methane emitted by farmed ruminants contributes 30.3% to New Zealand’s anthropogenic greenhouse gas inventory. Methanogens living in the rumen produce methane from H2 and CO2 as a byproduct of feed fermentation. The use of vaccines and small molecule inhibitors against the methanogens are promising methods to reduce methane emissions from extensively-grazed ruminants in New Zealand. Knowledge of the methanogens present in New Zealand ruminants is an important first step for successful vaccine and inhibitor development to target all methanogens. In this study, the methanogen diversity of farmed ruminants (sheep [Ovis aries], cattle [Bos taurus] and red deer [Cervus elaphus]) was investigated using molecular ecological techniques. Ruminants fed different diets had largely similar rumen methanogen communities. The major methanogen groups identified were from the Methanobrevibacter ruminantium clade (Mbb. ruminantium and closely-related species), Methanobrevibacter gottschalkii clade (Mbb. gottschalkii and closely-related species), Methanosphaera spp., and the putative methanogens belonging to the group designated Rumen Cluster C. A total of 37.5 - 57% of 16S rRNA genes in the rumen of a group of cows originated from members of Rumen Cluster C. Chloroform treatment of cows increased the abundance of Rumen Cluster C to 82% - 93% of archaeal 16S rRNA genes. In parallel, a total of 22% of mcrA genes belonged to an unassigned group of archaea, and chloroform treatment increased the unassigned group of archaea to 92% of all mcrA genes. This suggested that Rumen Cluster C archaea contain the gene mcrA. No members of the Rumen Cluster C group have previously been cultured, and currently there is no reported rumen isolate of Methanosphaera spp. A strain of Methanosphaera sp. was isolated from a sheep rumen and initial characterization suggests that this may be a new species. Three enrichment cultures were obtained containing members of Rumen Cluster C as the only archaea. Initial studies of these enrichment cultures showed that these three isolates were from three different sub-groups of Rumen Cluster C and that they produced methane. The investigation of methanogen diversity in New Zealand farmed ruminants and isolation of previously uncultured rumen methanogens reported here in this thesis will significantly aid the development of methane reduction strategies for farmed ruminants in New Zealand.