Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis. / Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis; however, the role of the peptide for the producing organismen is poorly understood. In this study we describe the first global approach to investigate this topic on a molecular level. Proteomic studies with M. aeruginosa PCC 7806 wild-type and several microcystin-deficient mutants indicated a physiological function for microcystin. Microcystin was shown to influence the abundance of several proteins which have an intra- or extracellular function. A prominent candidate is RubisCO, the key enzyme of the calvin cycle. RubisCO and other proteins, initially selected by 2D analysis, are putative cellular binding partners of microcystin. A potentially interaction mechanismen is the kovalent binding of microcystin to cysteine residues of the protein. Moreover, several knockouts of microcystin biosynthesis genes result in an overexpression of a putative morpho-type specific factor, named MrpC. This protein possibly mediates cell-cell interactions in Microcystis.

Made available in DSpace on 2016-06-02T19:29:50Z (GMT). No. of bitstreams: 1 TeseSPG.pdf: 896184 bytes, checksum: 0237c1553b20f09b9264cf38e782d113 (MD5) Previous issue date: 2004-06-30 / The influence of phytoplankton and their excreted materials in natural waters have been considered important issues among metal speciation studies. This work focused on the potential influence of the high molecular weight materials excreted by dominant phytoplankton species of Barra Bonita reservoir (Anabaena spiroides, Microcystis aeruginosa and Aulacoseira granulata) in the speciation and complexation of Cu and Cd. Therefore, this study comprised field and laboratory studies. Seasonal samplings were made during a period of two years in the reservoir. The total, ionic and organic concentrations of Cu and Cd were determined, as well as the complexation parameters with ligands present in the reservoir water. Also, the dissolved organic carbon and physico-chemical parameters of the water column were obtained. A speciation model was applied to metal complexation parameters and total metal concentrations obtained in field investigations. Laboratory cultures of the selected microalgae were made to obtain high molecular weight excreted materials. In a similar manner, the high molecular weight materials were isolated from the reservoir water. These materials were analysed for metal complexation with Cu and Cd, monosaccharide composition and elemental analysis (C, H, N and S). In the reservoir water, Cd speciation was dominated by ionic species, while Cu was mostly associated to the dissolved organic matter. Seasonal samplings presented Cu ligands with similar complexation parameters for the entire period studied. Cd complexation was not detected in the reservoir water. The speciation model resulted in similar Cu2+ concentration to the mean value obtained in field analyses, and indicated higher Cu association to the ligands of the reservoir water. Cu was complexed by all high molecular weight materials studied, while Cd was complexed solely by the cyanophyte materials. Higher proportions of acid monosaccharides in the high molecular weight materials corresponded to higher Cu and Cd complexation parameters, whereas no relation between C, H, N and S proportions and metal complexation properties of the high molecular weight materials was observed. The excreted materials of dominant microalgae from Barra Bonita reservoir had Cu ligands with intermediate to weak binding strength, as those present in the reservoir water. These results indicate the potential influence of such organic materials in metal speciation, bioavailability and mobility of metals with similar behaviour to that of Cu in the reservoir water. / A influência do fitoplâncton e de seus produtos excretados resultantes da fotossíntese em corpos d'água têm sido o enfoque de vários trabalhos que investigam a especiação e o destino de metais. Este estudo teve como hipótese a potencial influência dos materiais de alta massa molecular excretados por três espécies fitoplanctônicas (Anabaena spiroides, Microcystis aeruginosa e Aulacoseira granulata), dominantes no reservatório de Barra Bonita, em relação à especiação e complexação dos metais Cu e Cd na água do reservatório. Para tanto o estudo foi dividido em investigações de campo e experimentos de laboratório. Coletas sazonais por um período de dois anos no reservatório foram avaliadas quanto às espécies dissolvidas totais, orgânicas e iônicas de Cu e Cd, a determinação dos parâmetros de complexação destes metais com ligantes presentes na água, assim como o conteúdo de carbono orgânico dissolvido e os parâmetros físico-químicos da coluna d água. Uma modelagem da especiação química dos metais estudados foi feita com o uso das concentrações totais e os parâmetros de complexação adquiridos nas coletas sazonais. No laboratório foram obtidos os materiais de alta massa molecular excretados em cultivos das microalgas selecionadas, e o material de alta massa molecular dissolvido na água do reservatório, isolado seguindo-se o mesmo procedimento usado para os excretados algais. Estes materiais foram analisados quanto aos parâmetros de complexação com Cu e Cd, e composição monossacarídica e elementar (C, H, N e S). A especiação do Cd na água do reservatório foi dominada por formas iônicas, enquanto o Cu estava em grande maioria associado à matéria orgânica dissolvida. A água do reservatório apresentou, nas coletas sazonais, ligantes para Cu com parâmetros de complexação semelhantes em todo o período estudado. Nas condições experimentais empregadas, não foi detectada a complexação de Cd com ligantes oriundos da água do reservatório. A modelagem da especiação de Cu resultou em uma concentração semelhante de Cu2+ àquela medida em amostras de campo, e maior associação do Cu aos ligantes caracterizados neste estudo. O Cu foi complexado tanto pelos materiais de alta massa molecular das microalgas como também pela água do reservatório, enquanto o Cd foi complexado somente pelo material excretado pelas cianofíceas. Maiores proporções de monossacarídeos ácidos nos materiais corresponderam a parâmetros de complexação de Cu e Cd com valores mais elevados. No entanto, não foi observada uma relação entre as proporções de C, H, N e S e a complexação dos metais. Os excretados das microalgas dominantes no reservatório apresentaram ligantes para Cu com força de associação de intermediária a fraca, assim como os ligantes presentes no reservatório. Estes resultados confirmam o potencial de influência dos excretados na especiação, mobilidade e destino de metais com comportamento semelhante ao Cu, no reservatório de Barra Bonita.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work as main objective to evaluate the eletrogenic effect in preparations of Ãleum of rabbit fixes in chambers of Ãssing, in presence of supernatant of macrophages (S.MfS), stimulated with microcistin-LR (MCLR), of Microcystis aeruginosa. S.MfS estimulated with MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produces effect secretion, of the form dose-dependent; being that short circuit current (Isc), the secular chain variation (t) can be described for an equation of the type Isc = a . ekt for a correlation coefficient r = 0,9988 and Iscmaximo = 128,16  14,54 ÂA . cm-2. Later, was observed that the metabolic processes associatesâ geneses of the FSI, from stimulated macrophages, require the participation of a protein G, sensible pertussis active toxin. It was also verified that inhibing of protÃic synthesis, proteases, phosfolipase A2, ciclooxigenases, lipoxigenases, synthesis TNF-a, and antagonists of the PAF, they had reduced the FSI synthesis. With the application of monoclonal antibodies, it was verified that interleukin-b (IL-1b), was the main FSI; as also, that macrophages stimulated with MCLR, in the concentrations above, produced IL-1b and TNF-a, of form dose-dependent. The pay-treatment of the ileum mucosal with bumetanide, indomethacin, tetrodotoxin and HOE, disclosed that the secretory effect, by means of stimulated action of the S.MfS with MCLR is dependent of the secretion of ions chloride, with the participation of PAF, prostaglandins and mediators of the enteric nervous system. With this effect, it associates reduction of the transepitelial resistance (Rte), also mediated for prostaglandins, TNF-a, and indirectly IL-1b. The analysis of the coefficient of Hurst, disclosed that these effect had not occurred of random form, but had involved significant alterations in the kinetic parameters of the eletrogÃnic effect, to the level of the ileum mucosal. Macrophages stimulated for MCLR, produces and liberate more TNF-a of that IL-1b, to the Constant taxes, being this a linked characteristic to the type of employed stimulation. It is concluded, therefore, that MCLR stimulates macrophages to produce substances that can act as intestinal secretion factor, to the level of the ileum mucosal, involving chloride canals, reduction of the Rte, requesting for this, the participation of pro-inflammatory mediators and the enteric nervous system. / Este trabalho teve-se como principal objetivo, avaliar os efeitos eletrogÃnicos em preparaÃÃes de Ãleo de coelho fixadas em cÃmaras de Ãssing, em presenÃa de sobrenadante de macrÃfagos (S.MfS) estimulados com microcistina-LR MCLR de Microcystis aeruginosa. S.MfS estimulados com MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produz, de forma dose-dependente; sendo que a variaÃÃo temporal (t) da corrente de curto-circuito (Isc), pode ser descrita por uma equaÃÃo do tipo Isc = a . ekt; para um coeficiente de correlaÃÃo r = 0,9988 e Iscmaximo = 128,16  14,54 ÂÂcm-2. Posteriormente, observou-se que os processos metabÃlicos associados à gÃnese do fator deâ secreÃÃo intestinalâ (FSI), a partir de macrÃfagos estimulados, requer a participaÃÃo de uma proteÃna G sensÃvel à toxina pertusis ativa. Verificou-se tambÃm que inibidores de sÃntese protÃica, proteases, fosfolipase A2, cicloxigenases, lipoxigenases,; sÃntese de TNF-a e antagonista do PAF, reduziram a sÃntese de FSI. Com o emprego de anticorpos monoclonais, verificou-se que IL-1b era o principal FSI; como tambÃm, que macrÃfagos estimulados com MCLR, nas concentraÃÃes acima, reduziu IL-1b e TNF-a, de forma dose-dependente. O prÃ-tratamento da mucosa ileal com bumetanida, indometacina, tetrodotoxina e HOE, revelou que o efeito secretÃrio mediante aÃÃo do S.MfS estimulados com MCLR, à dependente da secreÃÃo de Ãons cloreto, com a participaÃÃo de PAF, prostaglandinas e mediadores do sistema nervoso entÃrico. A este efeito associa-se a diminuiÃÃo da resistÃncia transepitelial (Rte), tambÃm mediada por, prostaglandinas, PAF, TNF-a e, indiretamente, IL-1b. A anÃlise do coeficiente de Hurst (H), revelou que estes efeitos nÃo ocorreram ao acaso, mas envolveram alteraÃÃes significativas nos parÃmetros cinÃticos dos efeitos eletrogÃnicos, ao nÃvel da mucosa ileal. MacrÃfagos estimulados com MCLR produz e libera mais TNFa do que IL-1b, à taxas constantes ; sendo esta uma caracterÃstica atrelada ao tipo de estÃmulo empregado. Conclui-se, portanto, que MCLR estimula macrÃfagos a produzir substÃncias que podem atuar como fator de âsecreÃÃo intestinalâ ao nÃvel da mucosa ileal, envolvendo canais de cloreto, diminuiÃÃo de Rte; requisitando para isto, a participaÃÃo de mediadores prÃ-inflamatÃrios e do sistema nervoso entÃrico.

Considerando o aumento da ocorrência de florações de cianobactérias e a possibilidade de formação de subprodutos em diversos mananciais destinados ao consumo humano, este trabalho teve como objetivo, avaliar a remoção de células e subprodutos de Microcystis spp. pelos processos de dupla filtração com filtração ascendente em pedregulho e de filtração descendente em areia, oxidação e adsorção com carvões ativados pulverizado e granular. Na fase 1 foram realizados ensaios de bancada para estabelecimento das condições operacionais dos ensaios a serem realizados em instalação piloto, utilizando-se água de estudo preparada com adição de cultura de cepa tóxica de Microcystis spp. e ou extrato de microcistinas. Em seguida, foram realizados ensaios em instalação piloto de escoamento contínuo para águas de estudo preparadas com adição de cultura de cepa tóxica de Microcystis spp. e de extrato de microcistinas – fase 2 e de material coletado no reservatório de Barra Bonita – SP - fase 3. A concepção de dupla filtração utilizada mostrou ser bastante eficiente na remoção de células de Microcystis spp. e, consequentemente, de microcistinas intracelulares, porém, a remoção ou degradação de microcistinas extracelulares só foi significativa com o emprego de processos complementares de oxidação e ou adsorção. Para as águas de estudo empregadas, não houve formação expressiva de subprodutos organohalogenados nos efluentes dos processos de tratamento, após serem submetidas à cloração e tempo de contato de 1 dia. Adicionalmente, foram realizados estudos prospectivos no sentido de avaliar a toxicidade de extrato, águas de estudo e efluentes dos processos de tratamento por meio de bioensaios com cladóceros e camundongos, além de biomonitoramento com a espécie de peixe Danio rerio. / Considering the increase in the frequency of cyanobacterial blooms and the possibility of by-product formation in water supply sources for human consumption, this work was carried out in order to evaluate the removal of cells and by-products of Microcystis spp. using the processes of double filtration (gravel upflow filtration of coagulated water followed by rapid sand downflow filtration), oxidation and adsorption in powdered and granular activated carbons. Bench scale assays were performed in phase 1 to determine the operational conditions of the tests to be performed in a continuous flow pilot plant, using a study water prepared with filtered water in the water treatment plant 2 of Sao Carlos and culture of a toxic strain of Microcystis spp. and/or microcystins extract. Based on the conditions stated in phase 1, phase 2 was carried out by performing tests in the pilot plant for study waters prepared with culture of toxic strain of Microcystis spp. and phase 3 of microcystins extract from material collect in the de Barra Bonita – SP reservoir. The double filtration system used resulted very efficient concerning the removal of cells of Microcystis spp., but, on the other hand, the removal or degradation of extracellular microcystins was significant with the use of complementary processes such as oxidation and or activated carbon adsorption. With regards to the waters studied, there wasn’t significant formation of by-products due to the oxidation of the effluent of each combination of processes, when submitted to chlorination with detention time of 1 day. Additionally, it was observed the toxicity effect by bioassays with crustaceans and mices, besides Danio rerio fish screening.

In our pervious study, the ethanol extracts from Eupatorium fortunei Turcz at concentrations of 200 ÷ 500 mg L-1 significantly inhibited the growth of Microcystis aeruginosa, which is the most common species, responsible for toxic cyanobacteria blooming in fresh water. o-Coumaric acid (or 2-hydroxy-cinnamic acid) isolated from E. fortunei was tested its growth-inhibitory effect on M. aeruginosa and Chlorella vulgaris at the concentrations of 1.0, 10.0 and 100.0 mg L-1 in the 96- hour experiment by the optical density and the analytical method of chlorophyll a concentration. Results indicated that the compound strongly affected towards M. aeruginosa at the concentration of 100.0 mg L-1 with the inhibition efficiency (IE) values of 76.76 % and 84.66 %, respectively while those for C. vulgaris were lower just of 60.59%, and 74.53 %, respectively. The obtained data demonstrated that two methods were highly consistent and o- coumaric acid was more toxic to M. aeruginosa than C. vulgaris at all tested concentrations (p<0.05). The images of M. aeruginosa and C. vulgaris cells under the light microscope clearly showed the damage of these cells under the attck of o-coumaric acid. Although o-coumaric compound was widely demonstrated antibacterial properties in previous reports, to the best of our knowledge, our study was the first report about effect of o- coumaric acid on the growth of M. aeruginosa and C. vulgaris. / Những nghiên cứu trước đây đã chỉ ra rằng cao chiết etanol từ cây Mần tưới Eupatorium fortune Turcz tại dải nồng độ 200 ÷ 500 μg mL-1 ức chế mạnh sinh trưởng của Microcystis aeruginosa, - loài phổ biến nhất gây nên sự bùng nổ tảo độc trong hệ sinh thái nước ngọt. o-Coumaric axit (hay 2-hydroxy-cinnamic axit) phân lập từ E. fortune được tiến hành đánh giá ảnh hưởng lên sinh trưởng của hai loài M. aeruginosa và Chlorella vulgaris tại ba nồng độ là 1.0, 10.0 and 100.0 mg L-1 trong thời gian 96 giờ thực nghiệm theo phương pháp đo mật độ quang và phân tích hàm lượng chlorophyll a. Kết quả nghiên cứu cho thấy sau 96 giờ phơi nhiễm tại nồng độ 100 mg L-1 hoạt chất ức chế mạnh tới M. aeruginosa với giá trị ức chế sinh trưởng (IE) tương ứng là 76.76 và 84.66%. Giá trị IE đối với C.vulgaris ghi nhận thấp hơn chỉ là 60.65 và 74.53%, tương ứng. Hai phương pháp phân tích trên có tính nhất quán cao và o-coumaric ức chế sinh trưởng lên loài M.aeruginosa mạnh hơn so với loài C.vulgaris tại tất cả các nồng độ nghiên cứu (p<0.05). Ảnh chụp các tế bào M.aeruginosa và C.vulgaris dưới kính hiển vi điện tử đã chứng minh những tổn thương của tế bào dưới tác động của o-coumaric axit. Mặc dù o- coumaric cho thấy đặc tính chống khuẩn cao trong các công bố trước đây, nhưng theo hiểu biết của chúng tôi đây là nghiên cứu đầu tiên công bố về ảnh hưởng của hoạt chất o-coumaric axit lên sinh trưởng của M. aeruginosa và C. vulgaris.

Algae and cyanobacteria occur naturally in source waters and are known to cause extensive problems in the drinking water treatment industry. Cyanobacteria (especially Anabaena sp. and Microcystis sp.) are responsible for many water treatment problems in drinking water treatment works (DWTW) all over the world because of their ability to produce organic compounds like cyanotoxins (e.g. microcystin) and taste and odour compounds (e.g. geosmin) that can have an adverse effect on consumer health and consumer confidence in tap water. Therefore, the monitoring of cyanobacteria in source waters entering DWTW has become an essential part of drinking water treatment management. Managers of DWTW, rely heavily on results of physical, chemical and biological water quality analyses, for their management decisions. But results of water quality analyses can be delayed from 3 hours to a few days depending on a magnitude of factors such as: sampling, distance and accessibility to laboratory, laboratory sample turn-around times, specific methods used in analyses etc. Therefore the use of on-line (in situ) instruments that can supply real-time results by the click of a button has become very popular in the past few years. On-line instruments were developed for analyses like pH, conductivity, nitrate, chlorophyll-a and cyanobacteria concentrations. Although, this real-time (on-line) data has given drinking water treatment managers a better opportunity to make sound management decisions around drinking water treatment options based on the latest possible results, it may still be “too little, too late” once a sudden cyanobacterial bloom of especially Anabaena sp. or Microcystis sp. enters the plant. Therefore the benefit for drinking water treatment management, of changing the focus from real-time results to future predictions of water quality has become apparent. The aims of this study were 1) to review the environmental variables associated with cyanobacterial blooms in the Vaal Dam, as to get background on the input variables that can be used in cyanobacterial-related forecasting models; 2) to apply rule-based Hybrid Evolutionary Algorithms (HEAs) to develop models using a) all applicable laboratory-generated data and b) on-line measureable data only, as input variables in prediction models for harmful algal blooms in the Vaal Dam; 3) to test these models with data that was not used to develop the models (so-called “unseen data”), including on-line (in situ) generated data; and 4) to incorporate selected models into two cyanobacterial incident management protocols which link to the Water Safety Plan (WSP) of a large DWTW (case study : Rand Water). During the current study physical, chemical and biological water quality data from 2000 to 2009, measured in the Vaal Dam and the 20km long canal supplying the Zuikerbosch DWTW of Rand Water, has been used to develop models for the prediction of Anabaena sp., Microcystis sp., the cyanotoxin microcystin and the taste and odour compound geosmin for different prediction or forecasting times in the source water. For the development and first stage of testing the models, 75% of the dataset was used to train the models and the remaining 25% of the dataset was used to test the models. Boot-strapping was used to determine which 75% of the dataset was to be used as the training dataset and which 25% as the testing dataset. Models were also tested with 2 to 3 years of so called “unseen data” (Vaal Dam 2010 – 2012) i.e. data not used at any stage during the model development. Fifty different models were developed for each set of “x input variables = 1 output variable” chosen beforehand. From the 50 models, the best model between the measured data and the predicted data was chosen. Sensitivity analyses were also performed on all input variables to determine the variables that have the largest impact on the result of the output. This study have shown that hybrid evolutionary algorithms can successfully be used to develop relatively accurate forecasting models, which can predict cyanobacterial cell concentrations (particularly Anabaena sp. and Microcystis sp.), as well as the cyanotoxin microcystin concentration in the Vaal Dam, for up to 21 days in advance (depending on the output variable and the model applied). The forecasting models that performed the best were those forecasting 7 days in advance (R2 = 0.86, 0.91 and 0.75 for Anabaena[7], Microcystis[7] and microcystin[7] respectively). Although no optimisation strategies were performed, the models developed during this study were generally more accurate than most models developed by other authors utilising the same concepts and even models optimised by hill climbing and/or differential evolution. It is speculated that including “initial cyanobacteria inoculum” as input variable (which is unique to this study), is most probably the reason for the better performing models. The results show that models developed from on-line (in situ) measureable data only, are almost as good as the models developed by using all possible input variables. The reason is most probably because “initial cyanobacteria inoculum” – the variable towards which the output result showed the greatest sensitivity – is included in these models. Generally models predicting Microcystis sp. in the Vaal Dam were more accurate than models predicting Anabaena sp. concentrations and models with a shorter prediction time (e.g. 7 days in advance) were statistically more accurate than models with longer prediction times (e.g. 14 or 21 days in advance). The multi-barrier approach in risk reduction, as promoted by the concept of water safety plans under the banner of the Blue Drop Certification Program, lends itself to the application of future predictions of water quality variables. In this study, prediction models of Anabaena sp., Microcystis sp. and microcystin concentrations 7 days in advance from the Vaal Dam, as well as geosmin concentration 7 days in advance from the canal were incorporated into the proposed incident management protocols. This was managed by adding an additional “Prediction Monitoring Level” to Rand Waters’ microcystin and taste and odour incident management protocols, to also include future predictions of cyanobacteria (Anabaena sp. and Microcystis sp.), microcystin and geosmin. The novelty of this study was the incorporation of future predictions into the water safety plan of a DWTW which has never been done before. This adds another barrier in the potential exposure of drinking water consumers to harmful and aesthetically unacceptable organic compounds produced by cyanobacteria. / PhD (Botany), North-West University, Potchefstroom Campus, 2015

Algae and cyanobacteria occur naturally in source waters and are known to cause extensive problems in the drinking water treatment industry. Cyanobacteria (especially Anabaena sp. and Microcystis sp.) are responsible for many water treatment problems in drinking water treatment works (DWTW) all over the world because of their ability to produce organic compounds like cyanotoxins (e.g. microcystin) and taste and odour compounds (e.g. geosmin) that can have an adverse effect on consumer health and consumer confidence in tap water. Therefore, the monitoring of cyanobacteria in source waters entering DWTW has become an essential part of drinking water treatment management. Managers of DWTW, rely heavily on results of physical, chemical and biological water quality analyses, for their management decisions. But results of water quality analyses can be delayed from 3 hours to a few days depending on a magnitude of factors such as: sampling, distance and accessibility to laboratory, laboratory sample turn-around times, specific methods used in analyses etc. Therefore the use of on-line (in situ) instruments that can supply real-time results by the click of a button has become very popular in the past few years. On-line instruments were developed for analyses like pH, conductivity, nitrate, chlorophyll-a and cyanobacteria concentrations. Although, this real-time (on-line) data has given drinking water treatment managers a better opportunity to make sound management decisions around drinking water treatment options based on the latest possible results, it may still be “too little, too late” once a sudden cyanobacterial bloom of especially Anabaena sp. or Microcystis sp. enters the plant. Therefore the benefit for drinking water treatment management, of changing the focus from real-time results to future predictions of water quality has become apparent. The aims of this study were 1) to review the environmental variables associated with cyanobacterial blooms in the Vaal Dam, as to get background on the input variables that can be used in cyanobacterial-related forecasting models; 2) to apply rule-based Hybrid Evolutionary Algorithms (HEAs) to develop models using a) all applicable laboratory-generated data and b) on-line measureable data only, as input variables in prediction models for harmful algal blooms in the Vaal Dam; 3) to test these models with data that was not used to develop the models (so-called “unseen data”), including on-line (in situ) generated data; and 4) to incorporate selected models into two cyanobacterial incident management protocols which link to the Water Safety Plan (WSP) of a large DWTW (case study : Rand Water). During the current study physical, chemical and biological water quality data from 2000 to 2009, measured in the Vaal Dam and the 20km long canal supplying the Zuikerbosch DWTW of Rand Water, has been used to develop models for the prediction of Anabaena sp., Microcystis sp., the cyanotoxin microcystin and the taste and odour compound geosmin for different prediction or forecasting times in the source water. For the development and first stage of testing the models, 75% of the dataset was used to train the models and the remaining 25% of the dataset was used to test the models. Boot-strapping was used to determine which 75% of the dataset was to be used as the training dataset and which 25% as the testing dataset. Models were also tested with 2 to 3 years of so called “unseen data” (Vaal Dam 2010 – 2012) i.e. data not used at any stage during the model development. Fifty different models were developed for each set of “x input variables = 1 output variable” chosen beforehand. From the 50 models, the best model between the measured data and the predicted data was chosen. Sensitivity analyses were also performed on all input variables to determine the variables that have the largest impact on the result of the output. This study have shown that hybrid evolutionary algorithms can successfully be used to develop relatively accurate forecasting models, which can predict cyanobacterial cell concentrations (particularly Anabaena sp. and Microcystis sp.), as well as the cyanotoxin microcystin concentration in the Vaal Dam, for up to 21 days in advance (depending on the output variable and the model applied). The forecasting models that performed the best were those forecasting 7 days in advance (R2 = 0.86, 0.91 and 0.75 for Anabaena[7], Microcystis[7] and microcystin[7] respectively). Although no optimisation strategies were performed, the models developed during this study were generally more accurate than most models developed by other authors utilising the same concepts and even models optimised by hill climbing and/or differential evolution. It is speculated that including “initial cyanobacteria inoculum” as input variable (which is unique to this study), is most probably the reason for the better performing models. The results show that models developed from on-line (in situ) measureable data only, are almost as good as the models developed by using all possible input variables. The reason is most probably because “initial cyanobacteria inoculum” – the variable towards which the output result showed the greatest sensitivity – is included in these models. Generally models predicting Microcystis sp. in the Vaal Dam were more accurate than models predicting Anabaena sp. concentrations and models with a shorter prediction time (e.g. 7 days in advance) were statistically more accurate than models with longer prediction times (e.g. 14 or 21 days in advance). The multi-barrier approach in risk reduction, as promoted by the concept of water safety plans under the banner of the Blue Drop Certification Program, lends itself to the application of future predictions of water quality variables. In this study, prediction models of Anabaena sp., Microcystis sp. and microcystin concentrations 7 days in advance from the Vaal Dam, as well as geosmin concentration 7 days in advance from the canal were incorporated into the proposed incident management protocols. This was managed by adding an additional “Prediction Monitoring Level” to Rand Waters’ microcystin and taste and odour incident management protocols, to also include future predictions of cyanobacteria (Anabaena sp. and Microcystis sp.), microcystin and geosmin. The novelty of this study was the incorporation of future predictions into the water safety plan of a DWTW which has never been done before. This adds another barrier in the potential exposure of drinking water consumers to harmful and aesthetically unacceptable organic compounds produced by cyanobacteria. / PhD (Botany), North-West University, Potchefstroom Campus, 2015

This dissertation thesis focuses on the removability of algal organic matter (AOM) by coagulation during water treatment and also on the influence of AOM on the coagulation of other substances present in source water. Special emphasis is put on the description of coagulation mechanisms. The effectiveness of AOM removal by coagulation was investigated by coagulation tests performed with optimized doses of coagulants (aluminium or ferric sulphate) under different pH values. Peptides and proteins contained in cellular organic matter of cyanobacterium Microcystis aeruginosa were used in the experiments since they have been previously reported to disturb the coagulation process. Moreover, peptides and proteins underwent coagulation experiments together with kaolin particles, representing clay particles in turbid waters, in both the presence and absence of coagulants to investigate the effect of AOM on the coagulation of turbid waters. To enable the description of coagulation mechanisms, AOM were characterised in terms of charge, functional groups, molecular weight and ability to form dissolved complexes with coagulant metals. The experimental results demonstrated that the removability of peptides and proteins is greatly dependent on pH value and on the properties of the involved particles or molecules....

Microcistinas são potentes hepatotoxinas e promotoras de tumores encontradas em águas doces que causam riscos à saúde pública e, portanto, representam um sério problema para as estações de tratamento de água. O gênero de cianobactéria Microcystis é o mais conhecido produtor dessas toxinas e também o mais comumente encontrado formando florações em reservatórios de água usados para abastecimento público. Entretanto, algumas bactérias são capazes de utilizar essas toxinas como fonte de carbono o que pode contribuir para a sua remoção da água. Neste estudo foi avaliado o potencial de degradação da microcistina-XR (MCYST-XR) por bactérias heterotróficas isoladas de um sistema de abastecimento público de água da cidade de Piracicaba-SP. A cianotoxina MCYST-XR avaliada foi isolada da linhagem Microcystis aeruginosa NPLJ-4 e purificada. Culturas puras de 35 bactérias isoladas do sistema de abastecimento de água foram testadas. Para isso, cada bactéria foi inoculada em meio mínimo de sais contendo 60 g mL-1 de MCYST-XR purificada e após 144 horas a degradação da toxina foi avaliada por análise em LC-MS/MS. Os picos indicando a massa da microcistina-XR (m/z 1037) não foram detectados no meio de cultura de seis bactérias, as quais foram identificadas pelo sequenciamento quase completo do gene de RNAr 16S como pertencentes aos gêneros Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. e Acinetobacter sp. Este é o primeiro relato de uma linhagem de Acinetobacter capaz de degradar MCYST. A cinética de biodegradação da MCYST-XR mostrou redução de 80% em 72 horas, período em que as bactérias apresentaram o máximo crescimento. A presença do gene mlrA codificante da enzima microcistinase envolvida no metabolismo da microcistina foi avaliada nos genomas dos seis isolados bacterianos usando iniciadores de PCR específicos. Fragmentos de aproximadamente 800 pb foram amplificados em dois isolados, Bacillus sp.e Microbacterium sp. Ensaios de toxicidade utilizando o cládocero Daphnia similis foram realizados para verificar a efetiva remoção da MCYST-XR e a não formação de subprodutos tóxicos na via de biodegradacão. Os resultados negativos dos bioensaios após 48 horas indicaram a ausência de subprodutos tóxicos na via da degradação. / Microcystins are potent hepatotoxins and tumor promoters found in freshwaters that cause public health risks and thus represent a serious problem for water treatment plants. The cyanobacterium genus Microcystis is the most known toxin-producer and the most common bloom-forming in water reservoirs used for public supply. However, some bacteria are able to use these toxins as carbon source, which can contribute to its removal from water. This study assessed the potential for degradation of microcystin-XR (MCYST-XR) by heterotrophic bacteria isolated from a public water supply system of the city of Piracicaba-SP. The cyanotoxin MCYSTXR evaluated was isolated from Microcystis aeruginosa strain NPLJ-4 and purified. Pure cultures of 35 bacteria isolated from the water supply system were tested. For this, each bacterium was inoculated in minimal medium salts containing 60 g mL-1 of purified MCYST-XR and after 144 hours the toxin degradation was evaluated by LC-MS/MS analysis. The peaks indicating the mass of microcystin-XR (m/z 1037) were not detected in the culture medium of six bacteria, which were identified by almost complete sequencing of the 16S rRNA gene as belonging to the genera Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. and Acinetobacter sp. This is the first report of an Acinetobacter strain able to degrade MCYST. The kinetic of the MCYST-XR biodegration showed 80% reduction in 72 hours, period in which the bacteria showed the maximum growth. The presence of the mlrA gene encoding the microcystinase enzyme involved in the metabolism of microcystin was evaluated in the genomes of the six bacterial isolates using specific PCR primers. Fragments of approximately 800 bp were amplified in two isolates, Bacillus sp. and Microbacterium sp. Toxicity assays using the cladoceran Daphnia similis were conducted to verify the effective removal of MCYST-XR and the non formation of toxic by-products in the biodegradation pathway. The negative results of the bioassays after 48 hours indicated absence of toxic by-products in the biodegradation pathway.

Microcistinas são potentes hepatotoxinas e promotoras de tumores encontradas em águas doces que causam riscos à saúde pública e, portanto, representam um sério problema para as estações de tratamento de água. O gênero de cianobactéria Microcystis é o mais conhecido produtor dessas toxinas e também o mais comumente encontrado formando florações em reservatórios de água usados para abastecimento público. Entretanto, algumas bactérias são capazes de utilizar essas toxinas como fonte de carbono o que pode contribuir para a sua remoção da água. Neste estudo foi avaliado o potencial de degradação da microcistina-XR (MCYST-XR) por bactérias heterotróficas isoladas de um sistema de abastecimento público de água da cidade de Piracicaba-SP. A cianotoxina MCYST-XR avaliada foi isolada da linhagem Microcystis aeruginosa NPLJ-4 e purificada. Culturas puras de 35 bactérias isoladas do sistema de abastecimento de água foram testadas. Para isso, cada bactéria foi inoculada em meio mínimo de sais contendo 60 g mL-1 de MCYST-XR purificada e após 144 horas a degradação da toxina foi avaliada por análise em LC-MS/MS. Os picos indicando a massa da microcistina-XR (m/z 1037) não foram detectados no meio de cultura de seis bactérias, as quais foram identificadas pelo sequenciamento quase completo do gene de RNAr 16S como pertencentes aos gêneros Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. e Acinetobacter sp. Este é o primeiro relato de uma linhagem de Acinetobacter capaz de degradar MCYST. A cinética de biodegradação da MCYST-XR mostrou redução de 80% em 72 horas, período em que as bactérias apresentaram o máximo crescimento. A presença do gene mlrA codificante da enzima microcistinase envolvida no metabolismo da microcistina foi avaliada nos genomas dos seis isolados bacterianos usando iniciadores de PCR específicos. Fragmentos de aproximadamente 800 pb foram amplificados em dois isolados, Bacillus sp.e Microbacterium sp. Ensaios de toxicidade utilizando o cládocero Daphnia similis foram realizados para verificar a efetiva remoção da MCYST-XR e a não formação de subprodutos tóxicos na via de biodegradacão. Os resultados negativos dos bioensaios após 48 horas indicaram a ausência de subprodutos tóxicos na via da degradação. / Microcystins are potent hepatotoxins and tumor promoters found in freshwaters that cause public health risks and thus represent a serious problem for water treatment plants. The cyanobacterium genus Microcystis is the most known toxin-producer and the most common bloom-forming in water reservoirs used for public supply. However, some bacteria are able to use these toxins as carbon source, which can contribute to its removal from water. This study assessed the potential for degradation of microcystin-XR (MCYST-XR) by heterotrophic bacteria isolated from a public water supply system of the city of Piracicaba-SP. The cyanotoxin MCYSTXR evaluated was isolated from Microcystis aeruginosa strain NPLJ-4 and purified. Pure cultures of 35 bacteria isolated from the water supply system were tested. For this, each bacterium was inoculated in minimal medium salts containing 60 g mL-1 of purified MCYST-XR and after 144 hours the toxin degradation was evaluated by LC-MS/MS analysis. The peaks indicating the mass of microcystin-XR (m/z 1037) were not detected in the culture medium of six bacteria, which were identified by almost complete sequencing of the 16S rRNA gene as belonging to the genera Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. and Acinetobacter sp. This is the first report of an Acinetobacter strain able to degrade MCYST. The kinetic of the MCYST-XR biodegration showed 80% reduction in 72 hours, period in which the bacteria showed the maximum growth. The presence of the mlrA gene encoding the microcystinase enzyme involved in the metabolism of microcystin was evaluated in the genomes of the six bacterial isolates using specific PCR primers. Fragments of approximately 800 bp were amplified in two isolates, Bacillus sp. and Microbacterium sp. Toxicity assays using the cladoceran Daphnia similis were conducted to verify the effective removal of MCYST-XR and the non formation of toxic by-products in the biodegradation pathway. The negative results of the bioassays after 48 hours indicated absence of toxic by-products in the biodegradation pathway.