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Cyanobacterial blooms are of great concern to the drinking water treatment industry due to their capacity to produce microcystins and other cyanotoxins that are deadly to humans, livestock, pets, and aquatic life at low doses. Unfortunately, the strategies currently employed for cyanotoxin detection involve laborious analyses requiring significant expertise or bioassay kits that are subject to numerous false positives and negatives. These methods are incapable of providing rapid, inexpensive, and robust information to differentiate between the >80 cyanotoxin variants potentially present in an aqueous sample. The use of Raman spectroscopy for identification and quantification of the ubiquitous cyanotoxin microcystin-LR (MC-LR) was examined. Raman spectra readily reflect minute changes in molecular structure, spectra can be collected through water or glass, portable Raman spectrometers are increasingly available, and through surface enhanced Raman spectroscopy (SERS) it is possible to achieve femto or picomolar detection limits for a variety of target species. Drop coating deposition Raman (DCDR) was successfully implemented for quantitation of 2-100 ng of MC-LR deposited in 2 ?L of aqueous sample, even without the use of a specifically designed DCDR substrate or Raman signal enhancements. Reproducible MC-LR Raman spectra were observed for both fresh and aged DCDR samples, and the MC-LR Raman spectrum remained identifiable through a matrix of >80% DOM by mass. DCDR methods show tremendous potential for the rapid, simple, and economical detection of cyanotoxins in environmental matricies at environmentally relevant concentrations. / Master of Science

Sowohl in Süßwasserseen als auch in marinen Gewässern kommt es immer wieder zu Massenentwicklungen von Cyanobakterien, sogenannten “Blüten”. In Seen werden diese oftmals von Cyanobakterien der Gattung Microcystis dominiert, deren Arten häufig Toxine bilden. Die verbreitesten dieser Toxine sind die leberschädigen Microcystine, die eine Klasse nichtribosomal synthetisierter Peptide darstellen. Nachdem die toxische Wirkung der Microcystine bisher als deren Hauptfunktion angesehen wurde, deuten neuere Forschungsergebnisse darauf hin, dass Microcystine eine andere Primärfunktion für die Produzenten besitzen. Im Rahmen dieser Studie wurde Microvirin (Mvn), ein putatives Lektin aus Microcystis aeruginosa PCC 7806, von dem angnommen wurde, dass es funktional mit Microcystin assoziiert ist, charakterisiert. Zunächst konnte gezeigt werden, dass Mvn tatsächlich zuckerbindende Aktivität besitzt und spezifisch Mannan, ein Oligosaccharid aus Mannoseuntereinheiten, erkennt. Bindestudien zeigten, dass Zucker dieses Typs auf der Zelloberfläche von M. aeruginosa PCC 7806 lokalisiert sind und eine Bindestelle für das sekretierte Mvn darstellen. Mit Hilfe fluoreszenzmikroskopiebasierender Methoden wurde gezeigt, dass sowohl Mvn als auch das korrespondierende Mannanoligosaccharid stammspezifisch sind. Weiterhin konnte durch PCR gezeigt werden, dass das mvn-Gen in allen getesteten Microcystis-Stämmen vorkommt, die auch Gene für die Microcystinbiosynthese besitzen. Eine direkte Interaktion von Microcystin und Mvn konnte in vitro bestätigt werden. Microcystin bindet dabei über seinen N-Methyl-Dehydroalaninrest kovalent an die reduzierten Cysteinreste des Proteins. Ein Einfluss auf die Oligomerisierung des Proteins wurde festgestellt. Microcystin bindet an Cysteinreste von Proteinen, und es konnte gezeigt werden, dass dies besonders unter oxidativen Stressbedingungen geschieht. Die Daten liefern somit weitere Indizien für eine Rolle von Microcystin in der Stressadaptation. / Cyanobacteria frequently appear as so-called “water-blooms” during summer months. Cyanobacteria of the genus Microcystis, whose species often dominate freshwater lakes, produce toxins that represent a potential threat for humans and animals. The most prominent toxins are the non-ribosomally synthesised hepatotoxic microcystins. Toxicity has been considered the main function of these peptides, but recent studies propose different primary functions of microcystins for their producers. The involvement of microcystins in the response to oxidative stress was proposed recently. Within this study the putative lectin microvirin (Mvn), which was suggested to be functionally related to microcystin, was characterised. Initially it was shown that Mvn does indeed possess a carbohydrate binding activity, and specificity for mannan, an oligosaccharide made of mannose subunits, was proven. Binding studies using fluorescence-labelled Mvn and antibodies identified carbohydrates of this type at the cell surface of M. aeruginosa being a binding site for the secreted Mvn. Fluorescence microscopy techniques were employed to show that Mvn as well as the corresponding mannan oligosaccharide are strain-specific. Additionally it was shown by PCR that the mvn gene is present in all tested Microcystis strains possessing microcystin biosynthesis genes. A direct interaction of microcystin and Mvn was confirmed in vitro. Microcystin covalently binds to the reduced cysteine residues of the protein via its N-methyl-dehydroalanine moiety. An impact on the oligomerisation state of Mvn was observed. Microcystin seems to bind cysteine residues in an unspecific manner in vivo, and it was shown that this occurs especially under conditions of oxidative stress such as iron depletion and exposition to high light. Hence, the data provide further evidence for an involvement of microcystins in stress adaptation.

Cyanobacteria are one of the oldest forms of photosynthetic life and may have contributed significantly to the evolution of oxygen into the then anoxic environment. Cyanobacteria are also one of the best sources of natural secondary metabolites (cyanopeptides) some of which have harmful effects on the ecosystem, while others may be beneficial. It is known that these secondary metabolites are continuously produced during growth, however, it is not known whether the producing cyanobacteria actually benefit from these metabolites. The overarching aim of this study was to answer the question ‘Why do cyanobacteria produce secondary metabolites?’. With this aim in mind, preliminary work focused on understanding the growth and secondary metabolite production characteristics of Microcystis aeruginosa PCC7806. The technique of labelling secondary metabolites with 15N was successfully employed in differentiation and quantification of ex-novo and de-novo metabolites. The effect of exogenous cyanopeptides such as microcystins, aerucyclamides, anabaenopeptins, aeruginosamide, cyanopeptolin and aeruginosin on M. aeruginosa PCC7806 was evaluated using a rapid bioassay approach along with an automated cell enumeration technique. The results indicate that at least some cyanopeptides (microcystins-LR, microcystin-LF, aeruginosamide, anabaenopeptin B and aerucyclamide A) induce significant changes to cell division and metabolite production rate. In an ecological scenario, the release of such secondary metabolites by lysing cells (such as when blooms collapse), may be perceived as an alarm signal by surrounding live cells, which may in turn slow cell division and prepare for re-invasion. This may be a strategy for species survival and dominance. While the results from this study do not confirm a role for cyanopeptides, it is thought that the results are clearly indicative of the role played by cyanopeptides for the producing organism. In order to confirm a role, it is recommended that monitoring ribosomally synthesised metabolites (e.g. aerucyclamides) along with chlorophyll-a gene expression, with sophisticated techniques such as qPCR are used.

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.

A expansão agrícola e a urbanização têm agravado a eutrofização artificial dos mananciais superficiais, devido ao aumento de seus níveis de nutrientes, como nitrogênio e fósforo. Nesses episódios, o crescimento excessivo de cianobactérias de elevada capacidade adaptativa e potencial de produção de cianotoxinas pode prejudicar homens e animais. As microcistinas estão entre as cianotoxinas mais encontradas em florações de cianobactérias tóxicas, de difícil remoção pelas tecnologias convencionais de Estações de Tratamento de Água (ETA). Como barreira adicional no tratamento avançado de águas de abastecimento, destaca-se o processo de adsorção com carvão ativado pulverizado (CAP) ou granular (CAG). Esta pesquisa avaliou a eficiência de remoção de microcistina-LR (MC-LR) por oito carvões ativados (7 CAGs e 1 CAP) produzidos a partir de matérias-primas diversificadas. Os carvões foram caracterizados quanto a umidade, teor de cinzas, pH, massa específica aparente, número de iodo, índice de azul de Metileno, coeficiente de desuniformidade, área superficial específica, volume de micro e mesoporos, análise elementar por Espectroscopia de Energia Dispersiva e fotomicrografia por Microscopia Eletrônica de Varredura. Avaliou-se a capacidade adsortiva dos carvões sobre a MC-LR por isotermas descritas pelos modelos matemáticos de Langmuir e Freundlich. Para tais ensaios, foram utilizadas amostras trituradas de carvão. Além disso, foram operadas quatorze colunas CAG em escala de bancada, de modo intermitente, com concentração inicial (Co) de MC-LR na faixa de 69 a 137 µg/L. A concentração de toxina foi estimada pelo método ELISA (Ensaio de Imunoadsorção Enzimática). Os resultados indicaram que as propriedades do CA são influenciadas por seu material de origem e também pelo seu modo de produção, e que tais propriedades têm reflexo direto sobre a eficiência de remoção de MC-LR. Nos ensaios de adsorção de MC-LR com as amostras trituradas, os dados se ajustaram melhor ao modelo de Langmuir. O carvão à base de linhito (CGLIN), em dosagem de 100,0 mg/L e com 4h de tempo de contato, apresentou a maior capacidade de remoção (97,2%) de MC-LR (Co: 115,1 µg/L), com qe,máx de 10,6 mg/g. O bom desempenho do CGLIN foi associado ao seu maior volume de mesoporos (0,53 cm3/g). Correlação significativa foi observada entre volume de mesoporos e qe,máx (r = 0,98, Pearson), o que foi corroborado pela Análise de Componentes Principais. Nos ensaios nas colunas de CAG, o carvão de hulha, CGHU (Co: 85 g/L) apresentou o melhor desempenho, seguido pelo carvão de casca de coco, CGCO4 (Co: 137 µg/L), ambos com remoção média de MC-LR de 99,5%. Nos ensaios com as amostras trituradas, nenhuma das dosagens removeu MC-LR a concentrações menores que 1,0 µg/L – valor máximo permitido pela Portaria MS nº 2.914/11 para água potável – enquanto que no início do tratamento com as colunas de CAG, com exceção do carvão de coco CGCO1, o efluente das demais amostras foi compatível com este limite. A divergência de resultados entre os dois tipos de ensaios indica que os experimentos com amostras de CAG trituradas podem alterar sua capacidade adsortiva e induzir a uma escolha incorreta do carvão mais adequado. Com isso, os resultados oferecem subsídios para o entendimento do desempenho das CAs sobre a remoção de cianotoxinas, amparando a aplicação do processo de adsorção em ETAs para minimização da incidência de intoxicações por água contaminada. / The agricultural expansion and urbanization has aggravated the artificial eutrophication of surface water sources, due to the increase of nutrient levels, such as nitrogen and phosphorus. In these episodes, the excessive growth of cyanobacteria of high adaptive capacity and potential to produce cyanotoxins can harm humans and animals. Microcystins are among the cyanotoxins most found in toxic cyanobacteria blooms, difficult to remove by conventional technologies of Water Treatment Plants (WTP). As an additional barrier in the advanced water treatment, the adsorption process with pulverized (PAC) or granular activated carbon (GAC) is highlighted. This research evaluated the efficiency of microcystin-LR (MC-LR) removal by eight activated carbons (7 GACs and 1 PAC) produced from diversified raw materials. The carbons were characterized by moisture, ash content, pH, bulk density, iodine number, Methylene blue index, desuniformity coefficient, specific surface area, micro and mesopore volume, elemental analysis by Energy Dispersive X-Ray Detector and photomicrography by Scanning Electron Microscopy. The MC-LR adsorptive capacity of the carbons was evaluated by isotherms that follow the mathematical models of Langmuir and Freundlich. For these tests, carbon crushed samples were used. Besides that, fourteen GAC columns were operated on bench scale, intermittently, with initial concentration (Co) of MC-LR in the range of 69 to 137 µg/L. The toxin concentration was measured by ELISA (Enzyme-Linked Immunosorbent Assay). The results indicate that the AC properties are influenced by their source material and also by their production mode, and these properties have a direct effect on the efficiency of MC-LR removal. In the adsorption tests of MC-LR with the AC crushed samples, the data were better fitted to the Langmuir model. The lignite-based carbon (CGLIN), at a dosage of 100.0 mg/L and 4h of contact time, presented the highest MC-LR (Co: 115.1 µg/L) removal capacity (97.2%), with a qe,máx of 10.6 mg/g. The good performance of CGLIN was associated with its higher volume of mesopores (0.53 cm3/g). Significant correlation was observed between volume of mesopores and qe,max (r = 0.98, Pearson), which was corroborated by Principal Component Analysis. In the GAC columns tests, the coal CGHU (Co: 85 µg/L) presented the best performance, followed by the coconut carbon CGCO4 (Co: 137 µg/L), both with MC-LR average removal from 99.5%. In the trials with the crushed samples, none of the dosages removed MC-LR at values less than 1.0 µg/L – maximum permitted value by Portaria MS nº 2.914/11 for drinking water – while at the beginning of the treatment in the GAC columns, with the exception of the coconut carbon CGCO1, the effluent of the other samples was compatible with this limit. The divergence of results between these two types of assays indicates that the experiments with crushed samples may changes their adsorptive capacity and induce an incorrect choice of the most suitable AC. With this, the results provide support to understand the ACs performance on the cyanotoxins removal, supporting the application of the adsorption process in WTPs to minimize the incidence of poisoning by contaminated water.

Cyanobacteria produce a variety of secondary metabolites which possess amongst others antifungal, antibacterial, and antiviral properties. Being primary producers they are also a vital component within the food web. However, certain strains also produce toxic metabolites such as the hepatotoxins microcystin (MC) and nodularin (NOD). Their toxicity in combination with the increasing global occurrence has resulted in a drinking water guideline limit of 1 μg L-1 being issued by the World Health Organisation (WHO). However, these toxins are not only present in water, but can be accumulated by fish and shellfish. Currently, no regulations regarding cyanotoxin contaminated seafood has been established despite similar toxicity to routinely monitored marine toxins such as domoic acid (DA). To facilitate regular monitoring, a high performance liquid chromatography photo diode array (HPLC-PDA) analysis method for the detection of DA was optimised to enable the simultaneous detection of DA and nine cyanotoxins. This method was then utilised to determine cyanotoxin concentration in laboratory cyanobacteria strains. To assess the accumulation and depuration of cyanotoxins in the common blue mussel Mytilus edulis, three feeding trials were performed. During these, mussels were exposed to two cyanobacteria strains, Nodularia spumigena KAC66, Microcystis aeruginosa PCC 7813, both individually and simultaneously. A rapid dose dependent accumulation of cyanotoxins was observed with maximum concentration of 3.4 -17 μg g-1 ww accumulated by M. edulis, which was followed by a much slower depuration observed. During the final feeding trial, with N. spumigena KAC 66 and M. aeruginosa PCC7813, cyanotoxins were still detectable following 27 days of depuration. Mortality in all studies was 7% or less indicating that most mussels were unaffected by the maximum dose of 480 μg L-1 NOD (feeding study 1), 390 μg L-1 MC (feeding study 2), or 130 μg L-1 total cyanotoxins (feeding trial 3), respectively. Mortality in negative control tanks was lower throughout all three feeding trials ( < 1 - 2.6%). Consumption of a typical portion size (20 mussels) would result in ingestion of cyanotoxins at levels significantly higher than the WHO recommended tolerable daily intake (TDI) of 2.4 μg NOD and/or MCs for a 60 kg adult. This value was exceeded not only during the exposure period (maximum levels 270 - 1370 μg cyanotoxins per 20 mussels), but also at the end of the depuration period 39-600 μg cyanotoxins per 20 mussels. These results illustrated that cyanotoxin monitoring of seafood should be considered not only during, but also following bloom events. In an attempt to investigate the cyanotoxin budget of the experimental system, not only mussels, but cyanobacteria cultures, the tank water, and the mussel faeces were also analysed for their cyanotoxin content. Results showed that large quantities of MCs and NOD were unaccounted for during all exposure trials. The combined effect of cyanotoxin metabolism in M. edulis, biotic and/or abiotic degradation, protein binding, and losses during the extraction and analysis were thought to have contributed to the unaccounted cyanotoxin fraction. Mussel flesh was analysed for the presence of glutathione or cysteine conjugates, however, there was no evidence of their occurrence in the samples tested. Due to these discrepancies in the toxin budget of the system, the introduction of correction factors for the analysis of cyanotoxins in M. edulis was suggested in order to protect the general public.

Um dos bens mais preciosos da Humanidade é a água. Embora ela seja abundante no planeta, grande parte é imprópria para o consumo humano. E, devido ao crescimento populacional intenso, à concentração urbana e à poluição dos corpos d\'água superficiais e subterrâneos, a quantidade de água em condições para consumo vem se reduzindo em taxas alarmantes. Recentemente, um dos tipos mais comuns de contaminação dos corpos d´água tem sido a presença de cianotoxinas. Atualmente, não existe um estado brasileiro que não tenha problemas com florações excessivas de algas e os correspondentes transtornos causados às concessionárias que operam as estações de tratamento. Tem-se apontado que esse fato é devido principalmente ao aporte de nitrogênio e fósforo derivado do uso indiscriminado de detergentes e fertilizantes. O presente estudo tem como objetivo investigar a aplicação da fotocatálise heterogênea solar (TiO2 como fotocatalisador) na destruição da [D-Leu]-Microcistina-LR, potente toxina de ampla ocorrência nas florações de cianobactérias. A [D-Leu]-Microcistina-LR foi extraída de uma cultura de Microcystis &aelig;ruginosa. Foi utilizado um reator de placa plana de vidro recoberta com TiO2 para os estudos de degradação. Além disso, a irradiância durante os experimentos solares foi medida com o uso de espectrorradiômetro. Após os ensaios de degradação, a concentração da toxina foi determinada por CLAE. A cinética de mineralização da solução tratada foi determinada através de análises de COT. A toxicidade aguda e crônica do efluente foi quantificada através de análises utilizando camundongos e ensaios in vitro de inibição da proteína fosfatase. A partir dos experimentos realizados, pôde-se observar que foi necessário um tempo de tratamento de 150 min para que a concentração da microcistina fosse reduzida aos valores exigidos por lei (de 10 para 1 &mu;g L-1). Outra constatação muito importante que pôde ser feita é que a fotocatálise heterogênea solar foi eminente destrutiva, não só para a toxina, mas também para os demais componentes do extrato e dos compostos de degradação gerados. Obteve-se uma remoção de carbono de aproximadamente 90% com 90 min de exposição ao Sol. Além disso, os testes de toxicidade utilizando camundongos mostraram que o efeito agudo causado pela amostras iniciais foi eliminado; entretanto, os testes utilizando a enzima fosfatase indicaram que podem ter sido formados subprodutos que produzam um efeito crônico em mamíferos. Os experimentos realizados apontam para a viabilidade do uso da fotocatálise heterogênea solar para o tratamento de águas contaminadas pela [D-Leu]-Microcistina-LR, não só devido à sua destruição, mas também à grande remoção de matéria orgânica que pode ser alcançada. / One of the most precious assets of mankind is the water. Although it is abundant in the planet, the majority of it is not suited for human consumption. Due to the intense population growth, to the urban concentration, and to the pollution of surface water bodies and ground water, the amount of freshwater is decreasing at alarming rates. Recently, a common type of water bodies\' contamination is the presence of cyanotoxins. Presently, all over Brazil there are records of excessive algae blooms, with the corresponding nuisances caused to the companies that run water treatment stations. This fact is thought to be a result of nitrogen and phosphorus inputs due to the indiscriminate use of detergents and fertilizers. The purpose of the present study is to investigate the use of solar heterogeneous photocatalysis (TiO2 as the photocatalyst) for the destruction of [D-Leu]-Microcystin-LR, powerful toxin of widespread occurrence within cyanobacteria blooms. The [D-Leu]-Microcystin-LR was extracted from a culture of Microcystis &aelig;ruginosa. It was used a flat plate glass reactor covered with TiO2 for the degradation studies. The irradiance was measured during the experiments with aid of a spectrum radiometer. After the degradation experiments, the toxin concentration was determined by HPLC. The mineralization kinetics was determined by TOC analyses. The acute and chronic toxicities were quantified using mice and phosphatase inhibition in vitro assays. From the performed experiments, it was determined that 150 min are necessary to reduce the toxin concentration to the value demanded by law (from 10 to 1 &mu;g L-1). Another important finding is that the solar heterogeneous photocatalysis was really destructive process, not only for the toxin, but also for the other extract components and degradation products generated. A mineralization degree of 90% was achieved with 90 min of exposure to the sun. Moreover, toxicity tests using mice have shown that the acute effect caused by the initial sample was removed. However, tests using the phosphatase enzyme indicated that it may be formed products capable of inducing chronic effects on mammals. The performed experiments indicate the feasibility of using solar heterogeneous photocatalysis for treating contaminated with [D-Leu]-Microcystin-LR, not only due to its destruction, but also to the significant removal of organic matter that can be achieved.