Thesis (PhD Microbiology and Plant Pathology(Water resource management))-University of Pretoria, 2006. / Summary in English. Includes bibliographical references. Available on the Internet via the World Wide Web.

D.Phil. (Chemistry) / Please refer to full text to view abstract

Freshwater resources are threatened by the presence and increase of harmful algal blooms (HABs) all over the world. The HABs are sometimes a direct result of anthropogenic pollution entering water bodies, such as partially treated nutrient-rich effluents and the leaching of fertilisers and animal wastes. Microcystis species are the dominant cyanobacteria (algae) that proliferate in these eutrophic waters. The impact of HABs on aquatic ecosystems and water resources, as well as their human health implications are well documented. Countermeasures have been proposed and implemented to manage HABs with varying levels of success. These control measures include the use of flocculants, mechanical removal of hyperscums and chemical algicides. The use of flocculants such as PhoslockTM is effective in reducing the phosphates in a water body thus depriving nutrients that are available to cyanobacteria. The mechanical option entails the manual removal of hyperscums thus reducing the numbers of cyanobacteria cells that may be the inoculum of the next bloom. The major disadvantage of these two measures is cost. Copper algicides have been used successfully to control HABs in raw water supplies intended for potable purposes. The major disadvantages are copper toxicity and release of microcystins from lysed cyanobacteria cells. Algicides accumulate in the sediments at concentration that are toxic to other aquatic organisms and may also cause long-term damage to the lake ecology. In some studies, microcystins have been implicated in the deaths of patients undergoing haemodialysis. Therefore there is an increasing need to reduce the use of chemicals for environmental and safety reasons. Thus, the development of environmentally friendly; safe non-chemical control measures such as biological control is of great importance to the management of HABs. Some papers, describe bacteria, which were isolated from eutrophic waters, such as Sphingomonas species with abilities to degrade microcystins and Saprospira albida with abilities to degrade Microcystis cells. Further research is required to evaluate whether these bacteria are antagonistic towards cyanobacteria. Ideally, a combination of strategies should be introduced; that is, combine predatory bacteria that directly lyse the cyanobacteria with microcystin degrading bacteria that then ‘mop up’ the released microcystins. The major objective of this study was to isolate organisms that have a similar antagonistic properties; determine their mechanism of action and then develop a model to account for the interaction between the predator and prey as the basis for the development of a biological control agent. During the screening for lytic organisms from eutrophic waters of Hartbeespoort dam, seven bacterial isolates were obtained. Based on electron microscope observation, two of the isolates were found aggregated around unhealthy Microcystis cells. These were identified as Pseudomonas stutzeri strain designated B2 and <i.Bacillus mycoides strain designated B16. Based on efficiency and efficacy experiments B. mycoides B16 was a more effective antagonist than P. stutzeri B2. Furthermore the <i.B. mycoides B16: Microcystis critical ratio was found to be 1:1 in 12 days. Thus altering the predator-prey ratio by increasing the predator bacteria numbers reduced the Microcystis lysis time to six days. The B. mycoides B16 managed to reduce the population of alive Microcystis cells by 85% under turbulent conditions and 97% under static conditions in six days. The increase in predator bacteria numbers coincided with a decrease in growth of Microcystis. The study on the interactions of Microcystis aeruginosa and Bacillus mycoides B16 indicated a series of morphological and ultrastructural changes within the cyanobacteria cell leading to its death. These are summarised in a conceptual model that was developed. The predatory bacteria, B. mycoides B16 attached onto the Microcystis cell through the use of fimbriae and or exopolymers. During this attachment the bacteria released extracellular substances that dissolved the Microcystis cell membrane and interfered with the photosynthesis process. The presence of numerous bacterial cells that aggregated around Microcystis cell provided a ‘shade’ that reduced the amount of light (hv) that reached the Microcystis cell. In response to these adverse conditions, the Microcystis cell did the following: It expanded its thylakoid system, the light harvesting system, to capture as much light as possible to enable it to carry out photosynthesis and it accumulated storage granules such as phosphate bodies, glycogen and cyanophycin and swollen cells. Other researchers have also reported the swelling phenomenon prior to cell lysis but did not account for what might be the cause. During the course of the lysis process the Microcystis cell underwent a transition stage that involved changes from alive (with an intact membrane) to membrane compromised (selective permeability), to death (no membrane) and eventual cell debris. Due to the depleted Microcystis cells, the B. mycoides B16 (non-motile, non-spore former) formed chains, i.e., exhibited rhizoidal growth in search of new Microcystis cells to attack. In conclusion, the present evidence in this study suggests that B. mycoides B16 is an ectoparasite (close contact is essential) in its lysis of Microcystis aeruginosa under laboratory conditions. These findings that B. mycoides B16 is a predatory bacterium towards Microcystis aeruginosa need to be further evaluated under field conditions in mesocosm experiments (secluded areas in a lake) to determine the possibility of using this organism as a biological control agent. The study on the interactions of Microcystis aeruginosa and Bacillus mycoides B16 indicated a series of morphological and ultrastructural changes within the cyanobacteria cell leading to its death. These are summarised in a conceptual model that was developed. The predatory bacteria, B. mycoides B16 attached onto the Microcystis cell through the use of fimbriae and or exopolymers. During this attachment the bacteria released extracellular substances that dissolved the Microcystis cell membrane and interfered with the photosynthesis process. The presence of numerous bacterial cells that aggregated around Microcystis cell provided a ‘shade’ that reduced the amount of light (hv) that reached the Microcystis cell. In response to these adverse conditions, the Microcystis cell did the following: It expanded its thylakoid system, the light harvesting system, to capture as much light as possible to enable it to carry out photosynthesis and it accumulated storage granules such as phosphate bodies, glycogen and cyanophycin and swollen cells. Other researchers have also reported the swelling phenomenon prior to cell lysis but did not account for what might be the cause. During the course of the lysis process the Microcystis cell underwent a transition stage that involved changes from alive (with an intact membrane) to membrane compromised (selective permeability), to death (no membrane) and eventual cell debris. Due to the depleted Microcystis cells, the B. mycoides B16 (non-motile, non-spore former) formed chains, i.e., exhibited rhizoidal growth in search of new Microcystis cells to attack. In conclusion, the present evidence in this study suggests that B. mycoides B16 is an ectoparasite (close contact is essential) in its lysis of Microcystis aeruginosa under laboratory conditions. These findings that B. mycoides B16 is a predatory bacterium towards Microcystis aeruginosa need to be further evaluated under field conditions in mesocosm experiments (secluded areas in a lake) to determine the possibility of using this organism as a biological control agent. The study on the interactions of Microcystis aeruginosa and Bacillus mycoides B16 indicated a series of morphological and ultrastructural changes within the cyanobacteria cell leading to its death. These are summarised in a conceptual model that was developed. The predatory bacteria, B. mycoides B16 attached onto the Microcystis cell through the use of fimbriae and or exopolymers. During this attachment the bacteria released extracellular substances that dissolved the Microcystis cell membrane and interfered with the photosynthesis process. The presence of numerous bacterial cells that aggregated around Microcystis cell provided a ‘shade’ that reduced the amount of light (hv) that reached the Microcystis cell. In response to these adverse conditions, the Microcystis cell did the following: It expanded its thylakoid system, the light harvesting system, to capture as much light as possible to enable it to carry out photosynthesis and it accumulated storage granules such as phosphate bodies, glycogen and cyanophycin and swollen cells. Other researchers have also reported the swelling phenomenon prior to cell lysis but did not account for what might be the cause. During the course of the lysis process the Microcystis cell underwent a transition stage that involved changes from alive (with an intact membrane) to membrane compromised (selective permeability), to death (no membrane) and eventual cell debris. Due to the depleted Microcystis cells, the B. mycoides B16 (non-motile, non-spore former) formed chains, i.e., exhibited rhizoidal growth in search of new Microcystis cells to attack. In conclusion, the present evidence in this study suggests that B. mycoides B16 is an ectoparasite (close contact is essential) in its lysis of Microcystis aeruginosa under laboratory conditions. These findings that B. mycoides B16 is a predatory bacterium towards Microcystis aeruginosa need to be further evaluated under field conditions in mesocosm experiments (secluded areas in a lake) to determine the possibility of using this organism as a biological control agent. / Thesis (PhD (Water Resource Management))--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18477号 / 農博第2077号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4861(農学部図書室) / 31355 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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A oferta de água vem se tornando cada vez mais diminuta à medida que a população, a indústria e a agricultura se expandem. A contaminação dos mananciais gerada pelo descarte de efluentes domésticos e industriais leva a eutrofização, processo pela qual grande aporte de nutrientes, particularmente fosfatos, leva ao crescimento excessivo de algas. As cianobactérias são microrganismos procariontes, que vivem nos ambientes mais adversos. A floração dessas algas quando presente em mananciais destinados ao consumo humano gera sérios problemas à saúde humana, pois algumas dessas algas podem gerar toxinas, conhecidas como hepatotoxinas, neurotoxinas e dermatotoxinas, de acordo com sua ação farmacológica. Dentre as hepatotoxinas encontramos a microcistina, um heptapeptídeo cíclico que pode levar à morte em horas ou dias. O objetivo desse estudo foi viabilizar a técnica HPLC, já proposta por outros autores, para quantificar microcistinas-LR em reservatórios de água, em escala empresarial para ser implementada em laboratórios de análise de águas. Para o desenvolvimento da técnica, foram utilizadas amostras de uma lagoa com floração de Microcystis. Para determinar a eficiência da técnica cromatográfica, foram realizados estudos com outro método, através do kit ELISA. Nessa etapa do trabalho, verificou-se que a técnica HPLC é mais sensível e viável para a quantificação das microcistinas. Nos experimentos realizados com a cromatografia líquida, observou-se que a coluna C-18 LiChrosorb (25 cm) 7 Sm utilizada no método, e o solvente metanol apresentaram grande influência nos resultados. À medida que os experimentos foram executados, verificou-se o decréscimo da sensibilidade da coluna. Os resultados foram satisfatórios apenas após a limpeza realizada na coluna, onde os padrões apresentaram uma curva de correlação igual a 0,92. Este fato leva a concluir que as colunas precisam ser renovadas para análises mais sensíveis, como no caso das microcistinas. As amostras extraídas com metanol apresentaram resultados relevantes, isto é, quanto maior a concentração de metanol utilizado na extração, maior a concentração de microcistina-LR obtida nos resultados, concluindo o metanol ser o solvente apropriado para a extração. Por fim, conclui-se que o método desenvolvido é viável, apresentando algumas dificuldades para sua implantação em escala empresarial. / The water supply has been decreasing more and more as the population, industry and agriculture expand. The contamination of the water sources generated by the domestic and industrial effluents discharges leads to the eutrophization, process where the large presence of nutrients, particularly phosphates, causes excessive increase of algae. The cyanobacteria are procaryote microorganisms which live in the most diverse environments. The florescence of the algae when present in sources directed to human consumption generates serious problems to the human health, for some of them may produce toxins known as hepatotoxins, neurotoxins and dermotoxins, according to their pharmacological action. Among the hepatotoxins, the microcystin, a cyclic heptapeptide that can lead to death in hours or days, is found. The objective of this study was to make feasible the use of the HPLC technique, already proposed by other authors, to quantify microcystins-LR in water reservoirs, in enterprise scale to be implemented in water analysis laboratories. In order to develop the technique, samples of water from a pond with Microcystis florescence were utilized. To evaluate the efficiency of the chromatographic technique, studies were performed with another method, through the ELISA kit. In this phase of the work, it was verified that the HPLC technique is the most sensible and viable for the quantification of the microcystins. It was observed, in the experiments performed with the liquid chromatography, that the column C-18 LiChrosorb (25 cm) 7 Sm utilized in the method and the methanol solvent presented great influence in the results. As the experiments were realized, the decrease of the sensibility of the column was verified. The results were satisfactory only after the column being cleaned, when the patterns presented a curve of correlation equal to 0.92. This fact leads to the conclusion that the columns need to be renewed for more sensible analysis, like in the case of the microcystins. The samples extracted with methanol presented relevant results, that is, the greater the concentration of the methanol utilized, the higher the concentration of microcystin-LR obtained in the results, leading to the conclusion that methanol was the solvent adequate to the extraction. Finally, it was concluded that the method developed is feasible, presenting some difficulties concerning its implantation in enterprise scale.

As cianobactérias atuam no tratamento de águas residuárias em lagoas de estabilização. Seu estudo torna-se importante tanto pela atuação no referido tratamento como na possível produção de toxinas e seus efeitos nos sistemas aquáticos e saúde da população. Protocolos moleculares para culturas axênicas ou naturais foram testados e adaptados para as amostras analisadas. Este trabalho teve como objetivo avaliar o método molecular de eletroforese em gel de gradiente desnaturante nas espécies de Microcystis (cianobactérias) em um sistema de lagoas de estabilização. Foram utilizadas as técnicas de Reação de Polimerização em Cadeia (PCR) e Eletroforese em Gel de Gradiente Desnaturante (DGGE) do gene RNAr 16S como marcador molecular na análise de cianobactérias no sistema de lagoas de estabilização de São Lourenço da Serra - SP. Este sistema é composto por tratamento primário (caixa de areia), fossa séptica seguida de um sistema australiano (lagoa anaeróbia seguida por uma lagoa facultativa) e na saída do sistema de lagoas um tanque de cloração. Na amostra ambiental realizada na lagoa facultativa obteve-se, através de exame microscópico, o predomínio das espécies Microcystis aeruginosa e Microcystis flos-aquae. Através da técnica molecular de DGGE foi obtido um padrão de aproximadamente 18 bandas com a amostra ambiental. / Cyanobacteria are active in wastewater treatment in stabilization ponds. The study of these microrganisms is of a great importance considering their role in the referred wastewater treatments as well as their potential to produce toxins and their effects in aquatic systems and public health. Molecular protocols for pure or natural cultures were tested and adapted for the collected environmental samples. This work had as objective to evaluate the molecular method of denatuirng gradient gel electrophoresis in the Microcystis species (cyanobacteria) in a system of stabilization ponds. The techniques Polimerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) of the gene 16S rRNA as molecular marker were used in the cyanobacteria analysis in the system of stabilization ponds of São Lourenço da Serra - SP. This system is composed by primary treatment (box of sand), septic tank followed by an Australian system (anaerobic pond proceeded by an facultative pond) and in the exit of the system a clorine tank. In environmental sample accomplished in the facultative pond it was obtained, as microscopic result, the prevalence of the species Microcystis aeruginosa and Microcystis flos-aquae. Through molecular techniques a pattern of approximately 18 bands with these environmental sample was obtained.

Por suas características fisiológicas, cianobactérias se adaptam rapidamente em sistemas eutrofizados e geralmente predominam na comunidade fitoplanctônica. O crescimento excessivo desses microrganismos resulta em dificuldades e acréscimo nos custos do tratamento de água para consumo humano. Além da rápida colmatação dos filtros devido à grande massa orgânica, atribuição de cor e sabor, cianobactérias podem se apresentar tóxicas, liberar na água metabólitos nocivos e também serem precursoras na formação de subprodutos. Assim, este trabalho teve por objetivo avaliar a eficiência de remoção da cianobactéria Microcystis sp por pré-oxidação com cloro, coagulação com sulfato de alumínio, filtração direta e pós-cloração em escala de bancada, baseando-se nos parâmetros de turbidez, contagem celular, distribuição de tamanho e contagem de partículas, carbono orgânico dissolvido e subprodutos organoalogenados. Para atingir tal objetivo foi necessária a realização de experimentos para determinação da dosagem e do tempo de contato de cloro livre e a construção de diagramas de coagulação-filtração. Paralelamente foi determinado o Potencial de Formação de Trialometanos. A presença de cianibactérias não pareceu favorecer a formação de subprodutos indesejados da cloração. O ensaio final foi realizado com pré-cloração, coagulação, filtração e pós-cloração. Para a água pré-clorada com 2,5 mg/L de cloro livre, coagulada com 4 mg/L de sulfato de alumínio comercial, filtrada em areia com tamanho de grãos entre 0,3 a 0,59 mm e pós-clorada com 3,0 mg/Lde cloro livre, a turbidez reduziu de 2,89 para 0,36 uT, redução de 88%. A contagem celular mostrou decréscimo de 99,98% e a contagem de partículas reduziu 96,3% na faixa de tamanho de 3 a 20 \'mü\'m. Também foi observado aumento do carbono orgânico dissolvido com o aumento da dosagem de cloro livre. / Because of their physiological characteristics, cyanobacteria are able to adapt themselves quickly in eutrophic systems and are usually predominant in water bodies. The excessive growth of these organisms results in an increase in water treatment costs and other problems. Beside faster filter clogging due to the large amounts of cyanobacteria, these organisms may be toxic, release toxic metabolites and act as nuisance subproducts precursors. The aim of this work is to evaluate Microcystis sp cyanobacteria removal efficiency by chlorine preoxidation, aluminum sulfate coagulation, direct filtration and post-chlorination in bench-scale experiments. The removal efficiency was evaluated based on turbidity, cell counts, particle size distribution, dissolved organic carbon and halogenated organic subproducts. To reach the objectives it was first necessary to do experiments to determine free chlorine dose and contact time and also prepare coagulation-filtration diagrams. The trihalomethane formation potential was also determined, showing the presence of cyanobacteria did not contribute to subproduct formation. The final test, involving prechlorination, coagulation, filtration and post-chlorination, showed excellent Microcystis sp removal results. For prechlorinated water with 2,5 mg/L of free chlorine, coagulated with 4 mg/L of aluminum sulfate, filtered in a sand bed with grain size between 0,3 and 0,59 mm and post-chlorinated with 3,0 mg/L of free chlorine, turbidity was reduced from 2,89 to 0,36 uT, a 88% decrease. Cell count showed a 99,98% decrease and the particle count was reduced in 96,3% in the size range of 3 to 20 \'mü\'m. An increase in dissolved organic carbon related to the increase in the free chlorine dose was also noted.

O aumento da incidência de florações de cianobactérias potencialmente tóxicas nos mananciais de abastecimento, favorecidas pelo elevado aporte de nutrientes nos corpos d\'água, compromete a qualidade da água de consumo e põe em risco a saúde humana e animal, além de elevar os custos do tratamento de água. A pré-cloração, tem se mostrado uma ótima opção tanto na inativação de cianobactérias como na remoção de cianotoxinas dissolvidas. No entanto, sob certas condições, pode causar lise celular e promover a liberação das toxinas no meio. O objetivo deste trabalho foi avaliar em escala laboratorial, o efeito da pré-cloração, utilizando como agente oxidante o hipoclorito de sódio, sobre a integridade celular de uma linhagem tóxica de Microcystis aeruginosa (LTPNA 08), por citometria de fluxo, e sobre a subsequente liberação e degradação das microcistinas (LR e RR) por LC-MS/ MS. Diferentes dosagens de cloro (0,05, 0,5, 1, 1,5, 2, 2,5, 3, 4 e 8 mg.L-1), tempos de contato (0, 15, 30 e 60 minutos) e densidade celular (1x106 células.mL-1 para os ensaios de jarros e 3,5 x106 células.mL-1 para o ensaio de viabilidade celular) foram utilizadas neste estudo. Os resultados obtidos nos ensaios de jarros mostraram remoções de microcistinas acima de 70% após 60 minutos de exposição ao oxidante, com 100% de remoção em doses de 2,5 e 3 mg Cl2.L-1. Valores de CT (concentração x tempo) acima de 40,66 mg.min.L-1 foram necessários para degradar as microcistinas a concentrações abaixo de 1,0 &#181;g.L-1, exigidos pela organização mundial de saúde (WHO) e pela legislação brasileira de potabilidade da água (Portaria MS nº 2914/2011). Não foi possível verificar a lise celular por microscopia óptica, no entanto, na análise por HPLC-DAD verificou-se degradação de mais de 70% da clorofila-a em todas as dosagens testadas, após 60 minutos de exposição, com a completa degradação nas concentrações de 2,5 e 3 mg.L-1 Cl2, indicando dano celular. Nos ensaios por citometria de fluxo, foi verificada a perda da integridade celular com o aumento da dosagem de cloro aplicada, observando-se a permeabilidade celular máxima, sem a desintegração da célula, na concentração de 2,5 mg.L-1 Cl2. Concentrações de 4 e 8 mg.L1 Cl2 promoveram a lise total das células, impossibilitando a permanência do marcador na célula. A perda dos pigmentos clorofila a e ficocianina ocorreram em concentrações de acima de 2,5 e acima de 1,5 mg.L-1 Cl2, respectivamente. O presente trabalho reforçou a eficiência da cloração na degradação das toxinas e os resultados obtidos podem ajudar as autoridades competentes a otimizar as práticas de cloração utilizadas no pré-tratamento da água. / The increased incidence of blooms of potentially toxic cyanobacteria in supply sources, favored by high input of nutrients in water bodies, compromises the quality of drinking water and affect human and animal health, besides increasing water treatment costs. The pre-chlorination, has proved a great choice both in the inactivation of cyanobacterial cells as in removing dissolved cyanotoxins. However, under certain conditions, can cause cell lysis and release toxins. The objective of this study was to evaluate in laboratory scale, the effect of pre-chlorination, using sodium hypochlorite, on cell integrity of toxic Microcystis aeruginosa (LTPNA 08) using flow cytometry, and the subsequent release and degradation of microcystins (LR and RR) by LC-MS / MS. Different chlorine doses (0.05, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 8 mg.L-1), contact times (0, 15, 30 and 60 minutes) and cell density (1x106 células.mL-1 for jar-test and 3.5 x106 células.mL-1 for cell viability assay) were used in this study. The results obtained in the jar- test showed degradations up to 70% after 60 minutes of exposure, with complete degradation at chlorine doses of 2,5 e 3 mg.L-1. Chlorine exposure (CT) values over 40,66 mg.min.L-1 were required for oxidation of microcystin LR and RR to concentrations below the World Health Organization (WHO) and Brazilian legislation for water potability (Portaria MS nº 2914/2011) guideline value of 1&#181;g.L-1. No differences in cell number was observed by microscopy, however, analysis by HPLC-DAD found chlorophyll-a reductions of more than 70% in all dosages tested after 60 minutes exposure, with values below the limit of quantification for concentrations of 2.5 and 3 mg.L-1 Cl2, indicating cell damage. In assays using flow cytometry, loss of cell integrity was observed with increasing chlorine concentration. The maximum cell permeability without cell disintegration was observed at a concentration of 2.5 mg.L-1 Cl2. Concentrations of 4 and 8 mg.L-1 Cl2 lead to complete cell lysis, making impossible the permanence of SYTOX Green in the cell. The loss of pigment chlorophyll a and phycocyanin occurred in concentrations above 2.5 and 1.5 mg.L-1 Cl2, respectively. This study reinforced the efficiency of chlorination in the toxins degradation and the results can help the water authorities to optimize the chlorination practices used in the pretreatment of water.