Thermophiles dominate deep subsurface aquifers and represent the most ancient life on the planet. In this project, the culturable diversity of thermophiles that inhabit the world’s largest freshwater geothermal aquifer, Australia’s Great Artesian Basin (GAB), were explored using culture-dependent techniques. These studies largely focused on four unique microbial mat communities that exist in the runoff channel of the New Lorne Bore (registered number 17263) at temperatures between 52 – 72 °C and mostly targeted the cultivation of iron(III)- reducing bacteria due to their considerable influence on subsurface processes and likely involvement in the corrosion of GAB bores and pipelines. During this project a number of novel and modified screening methods were developed including high-throughput microenrichments of thermophiles using Biolog (Biolog Inc., U.S.A.) and U-bottom deep-well (1 ml) microtiter plates (Sarstedt, Germany) amended with seldom tested energy substrates and varied terminal electron acceptors (TEA) such as iron(III), sulphate, vanadium(V) and molybdenum(VI). Phylogenetic analyses of the isolates recovered in this project detected more than 15 novel phylotypes thereby extending the known culturable diversity of the GAB microflora. Complete phenotypic characterisations were performed on 9 GAB thermoanaerobic isolates, which resulted in the descriptions of 8 novel organisms including 4 novel genera: Thermotalea metallivorans (Ogg & Patel, 2009b), Fervidicola ferrireducens (Ogg & Patel, 2009c), Sporolituus thermophilus (Ogg & Patel, 2009d) and Fervidicella metallireducens (Ogg & Patel, 2010); and 4 novel species: Caloramator australicus (Ogg & Patel, 2009a), Caloramator mitchellensis (Ogg & Patel, 2011a), Thermovenabulum gondwanense (Ogg et al., 2010), and Desulfotomaculum varum (Ogg & Patel, 2011c); and amendments to the genus Caloramator (Ogg & Patel, 2011a) [given in Appendices 1 – 8]. Included in these characterisation studies was a modified method for determining an organisms mol % of G + C content of DNA using TempliPhi (Amersham Biosciences, U.K.) genome amplification kits to prepare the high molecular weight (HMW) genomic DNA prior to thermal denaturation. When the phenotypic data resulting from the strain characterisations was combined with the enrichment results, a complex model of the microbial oxidation of organic matter in the GAB at elevated temperatures (50 – 70 °C) was constructed.