Phenotypic characterisation of members of the Lachnospiraceae family isolated from ruminants: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand

Microbial fermentation in the rumen employs the metabolic capacity of microorganisms to degrade lignocellulose from the diets consumed by ruminant animals. Advances in genomic, metagenomic and culture independent methods for studying microbiomes have caused a lag in the functional characterisation of isolated cultures. Moreover, understanding the interactions between microbes during rumen fermentation may help in producing strategies to improve animal productivity and address environmental impact issues such as enteric methane emissions. In this study it is demonstrated how cultured strains are required to accurately describe the functional traits of rumen bacteria. Members of the Lachnospiraceae family are one of the most abundant bacterial groups in the rumen, however, many of its isolated members are yet to be fully characterised or properly classified. In this study, the genomes of 45 Lachnospiraceae strains sequenced in the Hungate 1000 project were functionally annotated using the web-based annotation tool, Protologger. These predictions were then compared with phenotypic traits from the corresponding strains, uncovered using microscopy, carbon utilisation testing, and by analyses of short-chain fatty acid production, and headspace hydrogen. The results indicate how the genome can assist in the culturing and studying of rumen microorganisms but should not be solely relied on for the elucidation of functional traits.

Phenotypic characterisations of the 45 Lachnospiraceae strains revealed a preference for the resultant soluble components of cellulose degradation rather than hemicellulose. Starch and pectin were more readily fermented in comparison to cellulose and xylan. End product analysis revealed that the studied strains produce acetate, butyrate and propionate, products known to contribute to host health and nutrition. Ethanol, formate, lactate and less commonly succinate were produced as fermentation products demonstrating the potential of the strains to participate in interspecies metabolite transfers. A subset of the strains including members of the genera Lachnospira, Eubacterium and Oribacterium as well as unclassified Lachnospiraceae bacterium strains were shown to produce methanol from pectin degradation. End products of fibrolytic fermentation by the 45 Lachnospiraceae strains can potentially act as substrates for methanogenic archaea. The results of this study help to improve the knowledge surrounding the poorly studied Lachnospiraceae family and iii increases the overall utility of the Hungate 1000 culture collection. Additionally, the comparison between genotypic predictions and the phenotyping, accentuates the importance of culture-based studies, providing an incentive to continue cultivating representative strains from the rumen environment to clarify how various micr oorganisms are contributing to rumen fermentation.