Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-04-27T14:14:48Z No. of bitstreams: 1 PDF - Natássya Nyuska Cabral de Lima.pdf: 1817457 bytes, checksum: c20885f87efcfd4edcd9466b67b55e64 (MD5) / Made available in DSpace on 2016-04-27T14:14:48Z (GMT). No. of bitstreams: 1 PDF - Natássya Nyuska Cabral de Lima.pdf: 1817457 bytes, checksum: c20885f87efcfd4edcd9466b67b55e64 (MD5) Previous issue date: 2015-11-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The occurrence of cyanobacterial blooms in water sources used for public supply, is increasingly common. Some genera of cyanobacterial have species potentially producing cyanotoxins, which can affect human health by direct contact through the skin or by ingestion of contaminated water or food. Classified according to their pharmacological action, cyanotoxins, are known as hepatotoxins, neurotoxins and dermatotoxins. Among the hepatotoxins we found the microcystin, a cyclic heptapeptide which can lead to death in hours or days. During the treatment of water for human consumption it is important to consider a technique to remove intact cells of cyanobacteria, since the use of oxidizing agents promotes cell lysis which can cause the release of toxins into the water. The Activated Carbon (AC) is presented as one of the best alternatives to the removal of organic and inorganic compounds due to its high power of adsorption. Aimed the fulfillment the current drinkability ordinance, as the concentration of extracellular cyanotoxins the present study sought to evaluate on a bench scale, the microcystin-LR removal efficiency study of water by adsorption on Activated Carbon (AC). The water study of was prepared with addition of lysed cells Microcystis aeruginosa cultivation. Were evaluated different pH values and the results confirmed that MC-LR adsorption was more efficient at acidic pH close to 5,0. Kinetic studies of adsorption were performed, analyzed according to the models of pseudo-first and pseudo-second order, getting the best fit the model of pseudo-first order, and adsorption isotherms to determine the adsorptive capacity of CA in relation to microcystins. The data of the isotherms were modeled according to the Langmuir isotherm and Freundlich, with best fit to the Langmuir model.The analytical method used during research to determine the concentration of microcystin-LR, after completion of kinetic experiments and isotherm was the High Performance Liquid Chromatography Coupled with Mass Spectrometry (HPLC-MS). The adsorption by AC palm coconut shell proved to be an efficient process for the removal of MC-LR, since averages toxin removal efficiencies above 90% were observed. / A ocorrência de florações de cianobactérias, em mananciais utilizados para abastecimento público, é cada vez mais frequente. Alguns gêneros de cianobactérias possuem espécies potencialmente produtoras de cianotoxinas, que podem afetar a saúde humana pelo contato direto através da pele ou por ingestão de água ou alimento contaminado. Classificadas de acordo com sua ação farmacológica, as cianotoxinas, são conhecidas como hepatotoxinas, neurotoxinas e dermatotoxinas. Dentre as hepatotoxinas encontramos a microcistina, um heptapeptideo cíclico que pode levar a morte em horas ou dias. Durante o tratamento de água para consumo humano é importante considerar uma técnica que remova células intactas de cianobactérias, pois o uso de agentes oxidantes promove a lise celular a qual pode causar a liberação de toxinas na água. O Carvão Ativado (CA) se apresenta como uma das melhores alternativas para a remoção de compostos orgânicos e inorgânicos devido a seu alto poder de adsorção. Visando o cumprimento da portaria de potabilidade vigente, quanto à concentração de cianotoxinas extracelulares o presente trabalho buscou avaliar em escala de bancada, a eficiência de remoção de microcistina-LR da água de estudo por meio de adsorção em Carvão Ativado (CA). A água de estudo foi preparada com adição de cultivo de células lisadas de Microcystis aeruginosa. Foram avaliados diferentes valores de pH e os resultados confirmaram que a adsorção de MC-LR se mostrou mais eficiente em pH ácido próximo a 5,0. Foram realizados estudos cinéticos de adsorção, analisados de acordo com os modelos de pseudo-primeira e pseudo-segunda ordem, obtendo-se melhor ajuste ao modelo de pseudo- primeira ordem, e isotermas de adsorção para determinar a capacidade adsortiva do CA em relação às microcistinas. Os dados das isotermas foram modelados segundo as isotermas de Langmuir e Freundlich, com melhor ajuste ao modelo de Langmuir. O método analítico utilizado durante a pesquisa para determinação da concentração de microcistina-LR, após a finalização dos experimentos de cinética e isoterma foi o Cromatógrafo Líquido de Alta Eficiência Acoplado a Espectrometria de Massas (CLAE-EM). A adsorção por CA de casca de coco de dendê se mostrou um processo eficiente para a remoção de MC-LR, visto que eficiências médias de remoção da toxina acima de 90% foram observadas.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-04-27T14:30:56Z No. of bitstreams: 1 PDF - Aluízio Gonçalves da Silva.pdf: 2861910 bytes, checksum: fec467664bbab2d62bb600299f8e0d10 (MD5) / Made available in DSpace on 2016-04-27T14:30:56Z (GMT). No. of bitstreams: 1 PDF - Aluízio Gonçalves da Silva.pdf: 2861910 bytes, checksum: fec467664bbab2d62bb600299f8e0d10 (MD5) Previous issue date: 2015-11-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Algal blooms and toxic cyanobacteria in fountains intended for human supply has been reported in many countries in recent decades due to impacts and human actions in the most diverse ecosystems on the planet. The presence of lineages of cyanobacteria producing cyanotoxins has negative effects on water bodies in particular in the intendend to the public supply due to the harmful effect of these substances on human health and animals. In general, cyanotoxins are not removed by conventional purifying water technologies. This study aimed to evaluate the efficiency of advanced oxidation process with the Fenton reagent in the oxidation of microcystin-LR water for public supply using conventional treatment steps coagulation, flocculation and sedimentation. The study water was prepared by adding the lysed cell cultivation Microcystis aeruginosa dechlorinated with potable water. Oxidation steps were performed, coagulation, flocculation and sedimentation JarTest programmed with rapid mixing -1 time of 10 s, rapid mixing gradient of 1000 s , flocculation time 20 min, flocculation gradient -1 -1 30 s and sedimentation velocity 1,4 cm.min . Different pH ranges were tested (2,0, 4,5 and + 7,0) and different concentrations of the Fenton reagent in the proportion of 1/3 H2O/Fe . The obtained results showed the oxidation of microcystin-LR in the treatments with pH 2,0; 4,5; and 7,0. By scanning the ions in the mass spectrometer it was possible to identify the peaks of mass/charge (m/z) fragments of enabling confirmation of oxidation by the presence of oxidation by products. Furthermore, after sedimentation the Fenton reagent enables significant removal being treatment T2 in time of 15 minutes the best for include percentage removal of turbidity 0,07 uT, true color 2,61 uH, COD 7,7 mg.L and MC-LR 0,07 μg.L. / Florações de algas e cianobactérias tóxicas em mananciais destinados ao abastecimento humano tem sido relatado em vários países nas últimas décadas devido aos impactos e ações do homem nos mais diversos ecossistemas do planeta. A presença de linhagens de cianobactérias produtoras de cianotoxinas tem efeitos negativo nos corpos hídricos em particular nos destinados ao abastecimento público devido ao efeito nocivo dessas substâncias à saúde humana e de animais. Em geral as cianotoxinas não são removidas pelas tecnologias convencionais de potabilização de água. O presente trabalho buscou avaliar a eficiência do processo de oxidação avançada com o reagente Fenton na oxidação da microcistina-LR de água destinada ao abastecimento público utilizando as etapas de tratamento convencional de coagulação, floculação e sedimentação. A água de estudo foi preparada com adição de cultivo de células lisadas de Microcystis aeruginosa com água potável desclorada. Foram realizadas as etapas de oxidação, coagulação, floculação e sedimentação em JarTest programado com tempo de -1 mistura rápida de 10 s, gradiente de mistura rápida 1000 s , tempo de floculação 20 min, -1 -1 gradiente de floculação 30 s e velocidade de sedimentação de 1,4 cm.min . Foram testados diferentes faixas de pH (2,0, 4,5, e 7,0) e diferentes concentrações do reagente Fenton na 2+ proporção de 1/3 de H2O/Fe . Os resultados obtidos mostraram a oxidação da microcistina-LR nos tratamentos com pH 2,0; 4,5; e 7,0. Através da varredura dos íons no espetrômetro de massas foi possível identificar os picos de massa/carga (m/z) dos fragmentos possibilitando a confirmação da oxidação através da presença dos subprodutos da oxidação. Além disso, após a sedimentação o reagente Fenton possibilitou a remoção significativa sendo tratamento T2 no tempo de 15 minutos o melhor por abrangir percentuais de remoção de turbidez 0,07 uT, cor verdadeira 2,61 uH, COD 7,7 mg.L e MC-LR 0,07 µg.L.

Nanofiltration (NF) membrane technology is effective for removal of natural organic matter (NOM) and Disinfection By-Product (DBP) precursors from treated surface water (Allgeier et al., 1995, Chellam et al., 2000, Smith et al., 2002). However, there is a need to control other micropollutants, such as compounds released from algal blooms. In this research, the feasibility of using NF for removal of cyanobacterial exudates was evaluated as a polishing process for conventionally treated surface water. Screening tests were conducted to compare the performance of four NF membranes, Filmtec's NF90 and NF270, and Hydranautics's LFC1 and NTR7450, for removal of NOM and cyanobacterial exudates. The source water for the experiments was derived from Lake Manatee (FL) following full scale treatment by enhanced coagulation and dual media filtration. Water samples were amended with low levels of three cyanobacterial exudates: microcystin-LR, geosmin and 2-Methylisoborneol (MIB). The rapid bench scale membrane test (RBSMT) protocol was used to test NF at four recoveries of 50%, 70%, 85% and 95%. Bulk organics (TOC and UV254) and inorganics (conductivity, total and calcium hardness) were monitored along with other operating parameters during the setting and recovery tests. Spike tests were performed by spiking microcystin-LR (9.5 to 12.0 micro g/L), geosmin (45 to 220 ng/L) and MIB (45 to 225 ng/L). Three NF membranes (NF90, NF270 and LFC1) were effective for over 90% rejection of TOC and associated disinfection by-product formation potential (DBPFP). Due to NF treatment, the bromide:TOC ratio increased resulting in a shift towards higher levels of brominated DBPFPs. Similarly, these three NF membranes (NF90, NF270 and LFC1) were effective for removal of microcystin-LR to below the World Health Organization (WHO) guideline of 1 micro g/L. Only two of the NF membranes tested (NF90 and LFC1), were capable of removing geosmin and MIB to levels below the taste and odor threshold. These membranes removed greater than 92% of the geosmin and MIB. Based on these bench scale tests, further testing of NF on a pilot scale is warranted.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Cyanobacterial toxins present a real and growing threat to humans and animals due to the projected increases in algal blooms resulting from increasing global temperature and pollution. Wild animals, livestock, pet animals and humans can be poisoned from contaminated drinking water. With the discovery of cyanobacterial toxins present in nutritional supplements, a new concern looms over consumers with threats of neurotoxin and hepatotoxin related damage from exposure to these products. To this end, work on the development of a freeze dried algal reference material was pursued for future use in environmental and nutritional supplement analysis. The first stage of the project was to prepare needed calibration standards, starting with homoanatoxin a, a homologue of the highly neurotoxic anatoxin-a compound. The resulting reference material (RM-hATX) had a homoanatoxin-a concentration of 20.2 ± 0.7 ?M, and proved to be stable while stored at temperatures of 80°C. Reference samples for dihydro and epoxy analogues of anatoxin-a and homoanatoxin-a were then prepared by semi-synthesis. The second stage of the project was the development of new analytical methods for the anatoxins. A derivatization reaction in which dansyl chloride was coupled with a novel cleanup step produced anatoxin derivatives suitable for liquid chromatography (LC) with mass spectrometry (MS) or fluorescence detection (FLD). Limits of quantitation were 60 ng L-1 and 1.6 ?g L-1 for the developed LC-MS/MS and LC-FLD methods, respectively, with the limit of quantitation significantly better than that of a previously developed method for the underivatized toxins based on HILIC MS/MS. Quantitative results for anatoxins in various algal samples using all three methods of analysis of were compared and it was found that there were no significant differences between the three methods. Unfortunately, experiments showed that the various toxin analogues did not elicit equimolar responses in either LC-MS/MS or LC FLD, thus indicating the importance of having individual calibration standards for quantitative analysis. The LC-MS/MS and LC-FLD methods were paired with a previously developed method for the analysis of hepatotoxic microcystins to screen a small number of nutritional supplement samples for cyanobacterial toxins. Microcystins were detected in all five Aphanizomenon flos-aquae samples examined. This method involved a fifteen-fold pre-concentration using a solid phase extraction cartridge, which gave a 98% recovery of microcystins. The third phase of the project was the preparation and testing of a preliminary algal matrix reference material as a feasibility study for the eventual production of a CRM. After selecting various algal cultures and samples that could be blended together, a freeze dried algal reference material was prepared and packaged. This material (RM-BGA) was then characterized using several methods including the two new dansylation-based procedures.

Toxic cyanobacterial blooms constitute a threat to the safety and ecological quality of aquatic environments worldwide. Cyclic hepatotoxin, especially microcystin, is the most widely occurring of the cyanotoxins. The aim of this study was to identify the cyanobacterial genotypes present including how many toxic genotypes were present in two North American lakes and one African Lake. All three lakes are prone to cyanobacterial blooms and were sampled in 2005 and 2006: Lake Ontario (Bay of Quinte, Canada), Lake Erie (Maumee Bay, Canada) and Lake Victoria (Nyanza Gulf, Kenya). The cyanobacterial genotypic community was assessed using DNA based analyses of the hypervariable V3 region of the 16S rRNA gene. In addition, the aminotransferase (AMT) domain in modules mcyE and ndaF of the microcystin and nodularin gene cluster respectively was used to detect the presence of hepatotoxic genotypes. Denaturing gradient gel electrophoresis (DGGE) results from this study suggested that hepatotoxin producers were present in all study sites sampled and were most likely members of the genus Microcystis. This study was the first to report the potential for microcystin production in the in-shore and off-shore open lake of Nyanza Gulf in Kenya. A seasonal study of the Bay of Quinte and Maumee Bay showed differences in the cyanobacterial genotypic community from early to late summer. In addition, the cyanobacterial genotypic community from the Bay of Quinte differed from 2005 to 2006 and quantification of the North American samples revealed an increase in cyanobacterial cells from early to late summer. The Bay of Quinte saw relatively no change in hepatotoxic cells from early to late summer but in Maumee Bay hepatotoxic cells increased from undetectable in early summer to dominating the cyanobacterial community by late summer. This study demonstrated the use of DGGE and qPCR of the 16S rRNA-V3 and AMT gene region in monitoring the cyanobacterial community of waterbodies susceptible to toxic cyanobacterial blooms.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Les cyanobactéries sont des micro-organismes qui préoccupent les autorités de santé publique dans le monde entier, en raison de la toxicité des cyanotoxines qu'elles produisent. Certaines cyanotoxines dont les microcystines (MC) sont des hépatotoxines inhibitrices de protéines phosphatases à sérine/thréonine. Aujourd'hui, plus de 200 variants de MCs ont été identifiés. Il s'agit d'heptapeptides monocycliques synthétisés par voie non-ribosomale dont la MC-LR (cyclo- (D-Ala-L-Leu-D-érythro-β-méthylAsp-L-Arg-ADDA-D-Glu-N-méthyl-hydro-Ala) est le variant le plus étudié en raison de sa fréquence et de sa forte toxicité. L’objectif de cette étude est le développement d'une méthode d'immunoanalyse rapide, sensible et fiable pour détecter les MCs. Le projet vise donc à développer un outil alternatif de détection de la MC-LR, qui serait mieux adapté aux analyses sur le terrain que les méthodes analytiques, biologiques ou les méthodes d'inhibition d'activité enzymatique actuellement disponibles. L'originalité de ce projet réside dans l'utilisation de deux approches différentes pour sélectionner de nouveaux anticorps spécifiques de la MC-LR. La première repose sur l'immunisation d'animaux de laboratoire, la technologie d'hybridation cellulaire et la sélection d'hybridomes sécréteurs d'anticorps monoclonaux. Si la méthodologie mise en œuvre a effectivement permis d'obtenir des immun-sérums spécifiques, la sélection des hybridomes d'intérêt reste à optimiser. La seconde stratégie mise en œuvre est basée sur la technologie du phage display pour sélectionner des fragments d'anticorps spécifiques de MC-LR à partir d'une banque de taille d’environ 109 phages, exprimant en surface des anticorps sous un format scFv (Shahsavarian et al., 2014). Plusieurs méthodes de criblage ont été développées et trois scFv ont été sélectionnés et étudiés, parallèlement à un quatrième scFv identifié dans une étude précédente (McElhiney et al., 2002), tous spécifiques à la MC-LR. Ces scFv ont été produits sous forme libre, soluble et leur spécificité à la MC-LR a été évaluée par ELISA et résonance plasmonique de surface. Les résultats obtenus montrent que les scFv sélectionnés sont tous capables de reconnaître la MC-LR. Néanmoins, ces résultats sont peu reproductibles et remettent en question le protocole de renaturation utilisé. Un travail de fond sur l’optimisation du protocole de renaturation s’avèrerait nécessaire pour les scFv ici sélectionnés, afin d’identifier les paramètres précis aboutissant à la perte ou au gain de leur fonctionnalité. / Cyanobacteria are ubiquitous microorganisms that present a worldwide concern to public health authorities because of the toxicity of the cyanotoxins they produce. Some cyanotoxins are hepatotoxins such as microcystins (MCs). At least 200 variants of MCs have been identified till today. In our study, we focus on MC-LR, a monocyclic heptapeptide (cyclo-(D-Ala-L-Leu-D-erythro-β-methylAsp-L-Arg-ADDA-D-Glu-N-methyldehydro-Ala), since it is the most frequently detected and one of the most toxic. In our study, we are interested in developing a fast, sensitive and reliable method to detect MCs. The project aims to develop an alternative pollution detection method that would be better suited to field measurements than the physicochemical methods currently available. The originality of this project lies in the use of two different approaches to select a panel of antibodies suitable for the development of immunodetection tests. The first one is based on the hybridoma technology for the production of monoclonal antibodies. The second one is based on phage display technique to select antibody fragments that are specific to MC-LR from a library of approximately 109 phages, expressing on the surface scFv fragments (Shahsavarian et al., 2014). Two monoclonal antibodies were selected using the first approach, and their specificity was evaluated using ELISA technique. Along with three scFvs selected from phage display approach. An additional scFv was added to this list: 3A8, selected from a previous study (McElhiney et al., 2002) and also specific to MC-LR. The scFvs were cloned into an expression vector in order to get each clone in its scFv soluble form. Then, their specificity to MC-LR was evaluated using ELISA technique and Surface plasmon resonance. The results show a potential specificity to MC-LR. Nevertheless, these results are not very reproducible and call into question the refolding protocol used. A thorough work on this protocol optimization would be necessary, in order to find the key parameters that control the loss or gain of their functionality