Molecular assessment of the occurrence of toxic cyanobacteria and cyanotoxins in South African impoundments
Conradie, Karin Ronel
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The theme of this thesis is the ecophysiological basis for the development of mass occurrences of Microcystis species in South African impoundments. Research concerning the reasons for harmful algal bloom (HAB) formation has been intensifying over the past few decades and the need for molecular investigations on natural occurring HAB species has been emphasised in recent literature. The main objectives of this investigation were to: • Verify the identity of specific bloom forming species by 16S rDNA analyses and to investigate the eco-physiological rationale for the mechanisms influencing growth and toxin production of the cyanobacteria. In order to realize this objective we had to achieve the following aims: • To verify the identity of the reference cultures in the culture collection of the North-West University by means of 16S rDNA sequencing and analysis. • To measure the DNA copy number of the Microcystis specific 16S rDNA and microcystin producing genes, mcyB as well as mcyE in order to shed more light on toxin production in the sampled environmental water, as well as the occurrence of toxigenic strains. • To investigate the in vivo expression of the ntcA and the rbcL genes in order to study the physiological rationale for the sudden increase in biovolume of a specific species during a bloom. A polyphasic approach was used for the taxonomic identification of some of the bloom forming species grown in the culture collection of the North West University to be used as reference cultures during the study. It was demonstrated that the isolate formerly known as "Oscillatoria simplicissima" should be reassigned to Planktothrix pseudagardhii under the order Oscillatoriales, family Phormidiaceae and subfamily Phormidioideae. Other strains in the culture collection were also not correctly identified, e.g. Spirulina sp. was in fact Arthruspira sp. To obtain a reliable quantification method each real-time polymerase chain reaction (PCR) method had to be optimised. A reliable protocol for DNA isolation that does not discriminate between the different cyanobacterial species and cell types, and so influence results, was also developed. The regulation of the gene expression of key metabolic enzymes was investigated in the Hartbeespoort Dam and the Roodeplaat Dam during the bloom season of 2004 to 2005. The in vivo expression of the ntcA and the rbcL genes were examined. The expression of these genes, rbcL (encoding the large subunit of Rubisco) and ntcA (encoding a nitrogen assimilation regulatory protein), reflect in part the photosynthetic and nitrogen metabolism activity of the cyanobacteria present in the sample. Together with this, DNA copy number of the Microcystis sp. specific 16S rDNA and toxin genes, mcyE as well as mcyB was also measured with real-time PCR. The trends of the ecological and molecular data were analysed using multivariate statistical analysis. Although the Roodeplaat and the Hartbeespoort Dams are closely situated in the densely populated area of the Gauteng province in South Africa, and both are eutrophic water impoundments, they differ remarkably in their responses to environmental influences, most probably due to the difference in nutrient loading and water surface temperatures. A very important variable in both dams is the inflow of the water into the system, representing nutrient loading. This nutrient loading is the main reason for the bloom occurrence to take place. The inflow of nutrients constitutes mainly of phosphorus and nitrogen. Clear relationships emerged between the total nitrogen in the water and the Microcystis sp. biomass, indicating that the decrease in nitrogen concentrations was caused by the increase in Microcystis sp. biomass. The Hartbeespoort Dam's biomass consists of different Microcystis sp. strains, some of which are toxic and some that are non toxin producers. The biomass of the Roodeplaat Dam however, consists mainly of toxic Microcystis species, but other photosynthetic species are also present in the Roodeplaat Dam. Microcystin production is associated with higher temperatures, and the release of microcystin is most probably caused by cells that die off due to natural cycles or temperatures. An association was also observed between ntcA gene expression and microcystin synthesis. It is argued that the ntcA gene increased as a result of high microcystin concentrations in the water, and thus probably inhibited the synthesis of microcystin. The Water Research Commission (WRC) has recently launched a program for the modulation of the production of harmful algal blooms. The data from this study is the first step to identify specific strains to be used for modulation and with this study, the first year's data is already submitted. The information can then be applied by the industry to predict when toxic Microcystis sp. is going to form a bloom under certain conditions. Therefore water purification plants can prepare in advance for a blooming event lowering the risk of distributing water of low quality to the public.