Culture- and Molecular-based Studies of the Archaeal communities Present in the Forestomach Microbiomes of the Tammar Wallaby (Macropus eugenii) and Western Grey Kangaroo (Macropus fuliginosus)

Kangaroos and wallabies are herbivorous mammals, collectively known as macropods, which like ruminants, gain nutritional benefit from microbial hydrolysis and fermentation of plant material during its retention in the animal‟s forestomach. However, there is a widespread belief based on limited results that little or no methane is released as a byproduct of feed digestion by macropods. Host and/or microbiological characteristics may be responsible for this “low methane emission” phenotype; but at the start of my thesis project the study of the forestomach microbiome of these animals lacked detail and scope. It is widely appreciated that methane formation in anaerobic microbial habitats is governed by a specific group of archaebacteria (hereafter referred to as methanogenic archaea) and; despite some evidence that archaea are present in the macropod forestomach, the perception has developed that methanogenic archaea are absent or at low densities in the macropod forestomach. Therefore, the goal of my PhD research was to use a variety of cultivation-dependent and -independent approaches, including metagenomics, to examine the phylogenetic and metabolic characteristics of the archaea present in the macropod forestomach. Results presented here in Chapter 2 confirm the presence of methanogenic archaea in the forestomach microbiome of the Tammar wallaby (Macropus eugenii), but their abundance was between 100- and 1000-fold less than the numbers typically observed in ruminant livestock. Species identified in the Tammar wallaby forestomach were most similar to Methanobrevibacter gottschalkii, Methanosphaera stadtmanae, and a Thermoplasmatales-affiliated methanogen (TaLC) previously identified from gut environments, including the rumen. Also, seasonal changes in the predominant species were observed, between sampling dates with a Mbr. gottschalkii species dominant at the autumn sampling date and the TaLC species dominant from spring sampled animals. Axenic culturing of archaea from the macropod forestomach has been prolonged and technically difficult, indicative of unknown interactions with other microbes that are supportive of growth; along with culture preservation is also very challenging. In that context, Chapter 3 describes the isolation and axenic cultivation of a Mbr. gottschalkii-like species from the Tammar wallaby forestomach microbiome, which was initially typed as a methanogenic autotroph, like the type strain, Mbr. gottschalkii HOT. A limited amount of phenotypic work was produced for this isolate prior to its preservation, but unfortunately, the Methanobrevibacter isolate lost viability during repeated culture and I was unable to resuscitate the isolate from freezer stocks despite repeated attempts. In Chapter 3, I describe better success in terms of maintaining archaeal viability within an enrichment culture produced using a sample of forestomach contents from a Western Grey kangaroo (Macropus fuliginosus). The enrichment culture was shown to possess a single archaeon vi species most closely affiliated with the order Thermoplasmatales and was shown to form methane using methylated amines as substrates. An enrichment culture containing a single strain of this same archaeon and produced from bovine rumen samples has also been successfully established by my colleagues, Stuart Denman and Jagadish Padmanabha. Chapter 4 provides an overview of the “metagenomic” sequence data produced from these two enrichment cultures by next generation sequencing with a specific focus on the data produced for the two archaeal strains. My analysis of these data lead to my hypothesis that these archaea are restricted to heterotrophic growth and I confirmed this by using various inhibitors known to target key steps in autotrophic and heterotrophic methanogenesis. Chapter 5 provides an overview of archaeon metagenomic sequence data from Tammar wallaby forestomach digesta and shows no archaea further to those detected in phylogenetic gene libraries and culture studies were present. Reassessment of the archaeal sequence data also reclassified some of these sequences as bacteria because of the appearance of recently sequenced data being uploaded to public databases. Taken together, the results presented in my thesis show methanogens are present in the macropod forestomach, albeit at numbers lower than those typically encountered in ruminants; with a change of the dominant species over time. The phylogenetic affiliations of these archaea are also similar to those found in ruminants, and my research provides the first definitive evidence confirming that the Thermoplasmatales-affiliated clade is indeed a sixth order of methanogenic archaea in Nature. In addition to the first genomic information for any of these archaea presented in my thesis, I have confirmed via the examination of the metagenomic datasets produced from Tammar wallaby samples that the archaeal populations in these animals are not comprised of any additional “not yet classified” archaea.