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The characterisation of three hyperthermophilic archaea from New Zealand hot springs

Archaea are a unique group of microorganisms that have been isolated from extreme environments such as geothermally active areas, highly saline habitats and extremely acidic sediments. Their capacity to withstand extreme conditions, and even thrive under them, makes them interesting objects of research. The aim of this research was to isolate and characterise a variety of hyperthermophilic archaea from New Zealand hot springs. Three organisms were chosen for investigation. The coccoid isolate Wai21.S1 is a novel strain of the genus Thermococcus that was isolated from a hot spring at Waimangu Volcanic Valley. It is only the second described fresh-water isolate of this genus. Wai21.S1 is strictly anaerobic and strictly heterotrophic. The preferred carbon sources are starch, amylose and amylopectin. Elemental sulfur is not required and its presence does not enhance growth. There is no dependence on sodium chloride. Wai21.S1 grows optimally at 89°C and at pH 5.9. The 16S rRNA gene sequence shows a close relationship to T. zilligii. Wai21.S1 is proposed to be the type strain of a new species Thermococcus waimanguensis. The isolate Tok2 was found in a sample from a hangi pool at Tokaanu, Turangi and was obtained from an enrichment culture containing konjac glucomannan as carbon source. Tok2 belongs to the genus Pyrobaculum. It is a strictly anaerobic organism which growths mainly heterotrophically on yeast extract and tryptone. Autotrophic growth was observed on CO₂/H₂ with thiosulfate or elemental sulfur as electron acceptor. Tok2 is rod-shaped with spherical bodies frequently observed at one or both ends, and the centre of the cells. Branched cells were occasionally observed. Tok2 grows optimally at 93°C and can grow up to 101°C. The optimum pH is 6.7. Tok2 is capable of reducing nitrate. The 16S rRNA gene sequence shows the close relationship of Tok2 to two other Pyrobaculum-like isolates from Tikitere, New Zealand the preliminarily named “Pyrobaculum neutrophilus” and strain IsoX. Phylogenetic data suggests that the three New Zealand isolates are strains of Pyrobaculum organotrophum despite strong physiological evidence against it. Further studies will be carried out to clarify the taxonomy of the New Zealand Pyrobaculum strains. Tok1, a second organism found in the same enrichments culture as Tok2, could not be isolated and was therefore maintained in a consortium together with Tok2. Tok1 is a regular coccus which grows heterotrophically on glucomannan as sole carbon source. It is microaerotolerant and grows optimally at 98°C. The optimum pH is 7.3. Strain Tok1 is highly sensitive to yeast extract and sensitive to elemental sulfur. The 16S rRNA gene sequence which was obtained by cloning, shows that Tok1 is a member of the kingdom Crenarchaeota and is a representative of a novel genus. Phylogenetically strain Tok1 is positioned at the base of the order “Igneococcales”. Since strain Tok1 could not be purified, the novel species had to be described as a candidatus. The name Candidatus “Igneosphaera vetusta” was proposed. The enzyme responsible for the degradation of konjac glucomannan was characterised and partially purified. The β-endomannanase was produced by strain Tok1 and is extremely thermostable. It shows highest activity around 110°C and is still enzymatically active at 120°C. The optimum pH for activity was at 6. Besides the mannanolytic activity, it exhibits a low xylanase activity. The enzyme is cell wall bound and no activity was found in the culture supernatant.
Type of thesis
The University of Waikato
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