Philosophiae Doctor - PhD / The El Tatio geyser field (ETGF) is the largest known geothermal field in Chile, forming part of a wide spectrum of extremophilic habitats in the country. The ETGF is NaCl rich, with high concentrations of toxic elements such as Li, As and Cs, which are contributed mainly by volcanic activities in the region. Most previous studies in the area have focused on the geology and geochemistry for mining purposes, as well as on the search for geothermal resources for power generation. Very little is currently known about the composition of the microbial communities of the ETGF, which makes the study reported here of particular novelty.A metagenomic approach, involving the amplification of 16S rRNA gene phylogenetic markers from metagenomic DNA was used to investigate seven different sites within the geyser field. The sample sites were characterized by high temperatures (80-85 °C) and a
range of pH values (6.3-8). Various molecular methods, including clone library construction and PCR-DGGE analyses were used to target a wide range of microbial populations within the ETGF sites. Multivariate analysis was also applied to assess differences in the microbial diversity from different sites and to correlate microbial diversity with environmental conditions. Culture-dependent screening of novel nanoarchaeal species was also undertaken.These were coupled with PCR and other detection methods such as fluorescent in situ hybridization (FISH) to trace the presence of nanoarchaeal signals from enriched cultures.The results have shown that the ETGF encompasses a limited microbial diversity represented by only 30 dominant phylotypes, and most likely due to the toxic chemical content of the
geyser field. The microbial representatives identified were assigned to OTUs from archaeal,nanoarchaeal and bacterial taxonomic groups. The dominant microbial taxa included members of the Proteobacteria, Firmicutes, Aquificae, Actinobacteria, Euryarchaeota(Halobacteriales, Archaeoglobales), Crenarchaeota (Thermoproteales, Desulfurococcales),together with uncultured representatives of the bacteria, archaea and nanoarchaeota. Notably,representatives of mesophilic, thermophilic and hyperthermophilic taxonomic groups were all detected in ETGF samples. This is attributed to various factors such as temperature gradients
and dispersal mechanisms (e.g. natural forces such as rain and volcanic activities). Principal component analysis (PCA) showed significant differences (P < 0.05) in the microbial diversity of the ETGF samples, with principal components (based on the sequenced species from both 16S rRNA clone libraries and PCR-DGGE profiles) explaining up to 62.7% of variance. Furthermore, CCA showed that the differences in phylogenetic diversity were most influenced by temperature and salinity. This was also confirmed by the sequencing results,which showed that hyperthermophilic and haloarchaeal taxa were dominant in the ETGF sites. However, conductivity and pH were also found to contribute to variations in the microbial diversity of the experimental samples, with TDS (total dissolved solids) being a less influential factor. Attempts to generate nanoarchaeal-host co-cultures, and to recover
sufficient nanoarchaeal genomic DNA for fosmid and/or large insert cloning for comparative genome analysis, were unsuccessful.This study is the first to employ metagenomic approaches to analyse the microbial diversity of sites in the ETGF, and has expanded our knowledge of microbiota present in this geyser field.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:etd.uwc.ac.za:11394/4014 |
| Date | January 2012 |
| Creators | Galada, Ncebakazi |
| Contributors | Cowan, D. A. |
| Publisher | University of the Western Cape |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of the Western Cape |
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