The human gut microbiome is a complex microbial ecosystem residing in the lumen of our gastrointestinal tract. The type and amounts of microbes present in this ecosystem varies based on numerous factors, including host genetics, diet, and environmental factors. The human gut microbiome plays an important role in normal host physiological functions, including providing energy to colonocytes in the form of short-chain fatty acids. However, gut microbial metabolites have also been associated with numerous disease states. Current tools for analyzing the gut microbiome, such as high-throughput sequencing techniques, are limited in their predictive ability. Additionally, “-omic” approaches of studying the complex array of molecules, such as transcriptomics (RNA), proteomics (proteins), and metabolomics (previously identified physiologically active molecules), give important insight as to the levels of these molecules but do not provide adequate explanations for their production in a complex environment. With a better physiological understanding of why specific metabolites are produced by the gut microbiome, more directed therapies could be developed to target their production. Therefore, it is immensely important to study the specific bacteria that reside within the gut microbiome to gain a better understanding of how their metabolic actions might impact the host. Within this framework, this study aimed to better understand the production of secondary bile acid metabolites by bacterial in the gut microbiome. High levels of secondary bile acids are associated with numerous pathophysiological disorders including colon cancer, liver cancer, and cholesterol gallstone disease. In the current study, three bile acid metabolizing strains of bacteria that are known members of the gut microbiome were studied. A novel strain of Eggerthella lenta was identified and characterized, along with the type strain, for its ability to modulate bile acid and steroid metabolism based on the atmospheric gas composition. Additionally, it was shown that the oxidation of hydroxyl groups on primary bile acids by E. lenta C592 inhibited subsequent 7α-dehydroxylation by Clostridium scindens. The gene involved in the production of a Δ4,6-reductase enzyme, responsible for catalyzing two of the final reductive steps in the 7α-dehydroxylation pathway, was putatively identified and characterized in Clostridium scindens ATCC 35704. Lastly, the transcriptomic profile of Clostridium scindens VPI 12708 in the presence of numerous bile acids and steroid molecules was studied. These studies contribute significantly to the understanding of why specific bile acid metabolites are made by members of the gut microbiome and suggest ways of modulating their production.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

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