The development and use of molecular phylogenetic and microscopy methods to study thermophilic bioleaching cultures

Mikkelsen, D. (2004). The development and use of molecular phylogenetic and microscopy methods to study thermophilic bioleaching cultures PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Mikkelsen, D.
Thesis Title The development and use of molecular phylogenetic and microscopy methods to study thermophilic bioleaching cultures
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Sly, Lindsay
Total pages 317
Collection year 2005
Language eng
Subjects L
270300 Microbiology
780105 Biological sciences
Formatted abstract

Bioleaching is the microbial solubilisation of metal cations from low-grade sulfidic ores. This process is essentially driven by consortia of chemolithotrophic iron- and sulfur-oxidising Bacteria and Archaea ubiquitous in sites of mineral oxidation, and occurs over a pH range of 0.5 to 2.0. In this study molecular techniques were developed to monitor thermophilic Archaea in cultures leaching chalcopyrite (CuFeS2), a copper sulfidic ore, at a temperature of 78 o C. 


A molecular phylogenetic approach was followed to develop molecular methods based on 16S rRNA gene sequences of community members and reference cultures. Analysis of 16S rRNA gene clone libraries was used to investigate the microbial communities present in the thermophilic bioleaching consortia. These revealed that Archaea belonging to the order Sulfolobales dominated the cultures, forming five distinct monophyletic phylotypes, namely the Sulfurisphaera ohwakuensis-like (Phylotype I), Stygiolobus azoricus-like (Phylotype II), Sulfolobus shibatae-like (Phylotype III), Metallosphaera species J 1-like (Phylotype IV) and Acidianus infernus-like (Phylotype V) phylotypes. The microbial community present in the bioleaching culture adapted to tolerate a low concentration of copper ions (IC sample) appeared to be dominated by Sulfolobus shibatae-like microorganisms (69% of total clones). The adaptation of the bioleaching culture (IC sample) to tolerate an increase in copper ions (HT80 sample) caused a striking community shift. The thermophilic microbial community evolved to have Sf. ohwakuensis-like organisms (73.9% of total clones) as the predominant bioleachers in the presence of higher copper concentrations. Based on the 16S rRNA gene sequence similarities shared between these organisms and their respective closest known phylogenetic relatives, it is likely that the Sulfurisphaera ohwakuensis-like and Sulfolobus shibatae-like organisms represent new species within the order Sulfolobales


Five archaeal species-specific fluorescence in situ hybridisation (FISH) probes were designed targeting the monophyletic groupings identified in the 16S rRNA gene phylogenetic analysis. These probes were used in the in situ detection of the archaeal populations present in thermophilic bioleaching cultures. The results obtained largely supported the finding of the phylogenetic approach and highlighted the need to use additional molecular techniques, such as FISH, to confirm and validate the initial set of results obtained. 


Temperature gradient gel electrophoresis (TGGE) methods were developed to rapidly screen 16S rRNA gene phylotypes. TGGE profiles of 16S rRNA gene PCR products generated using total community DNA extracted from the thermophilic chalcopyrite bioleaching cultures also demonstrated that this technique was useful for determining the relative species composition of these samples. 


Finally, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) was used to extensively document the leaching of pyrite (FeS2), an iron sulfidic ore, by the pure cultures of Acidianus brierleyi, Sulfolobus metallicus and Metallosphaera sedula. The interactions of these microorganisms with pyrite ore particles were of particular interest in order to establish if the evidence suggested a dependence on close contact between the thermophiles and the ore surface. In addition, for all three cultures, the amount of iron in solution was monitored to provide quantitative evidence that the pyrite ore was being leached by the thermophilic microorganisms. The leach residues formed during pyrite leaching by the culture that appeared to be the most active leacher, Metallosphaera sedula, were characterised using energy dispersive x-ray microanalysis (EDX). The evidence from this study indicated that the thermophilic bioleaching microorganisms acted through the indirect bioleaching mechanism during the dissolution of pyrite.

Keyword Phylogeny -- Molecular aspects
Bacterial leaching
Additional Notes Development of molecular phylogenetic and microscopy methods to study thermophilic bioleaching cultures

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Fri, 24 Aug 2007, 18:44:28 EST