Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-10-22T16:15:38Z No. of bitstreams: 2 Tese - Rafael Silva Menezes - 2015.pdf: 4153954 bytes, checksum: d0df40aec34aafcfceb965189f3f940c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-23T10:14:27Z (GMT) No. of bitstreams: 2 Tese - Rafael Silva Menezes - 2015.pdf: 4153954 bytes, checksum: d0df40aec34aafcfceb965189f3f940c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-23T10:14:27Z (GMT). No. of bitstreams: 2 Tese - Rafael Silva Menezes - 2015.pdf: 4153954 bytes, checksum: d0df40aec34aafcfceb965189f3f940c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-27 / Financiadora de Estudos e Projetos- Finep / The microalgae biomass, mainly rich in lipids, carbohydrates and other compounds groups, has been considered as a promising raw material for biodiesel production. Despite the potential and advantages, there are technological barriers that prevent microalgae biodiesel production on a large scale. This research aimed to develop innovative technical and scientific studies to assess the feasibility of using freshwater microalgae as a raw material source for biodiesel production. This study allowed evaluating the biodiesel production from microalgae biomass, from the selection of strains, the production and biodiesel quality analysis, and thus enabling to raise important parameters on the cultivation and production of microalgae biodiesel. This research identified a freshwater microalga with potential for biodiesel production - Choricystis minor var. minor - which has content and fatty acid profile suitable for biofuel production, providing more biodiesel (115%) than soybean, by biomass direct transesterification. In studies of culture medium, was observed that the WC medium is the best for biodiesel production. The BBM medium is most suitable for pigments production, such as chlorophyll and carotenoids. Scale pilot production of Choricystis and microalgal biomass conversion into biodiesel showed that this microalgae presents volumetric productivity about 11 times greater than soybeans, with production estimated at 6 tonnes of biodiesel per hectare per year. A few adjustments are required for produced biodiesel quality parameters can achieve the specification adopted by the ANP. Thus, can be concluded that the biodiesel production using microalgae can become a reality on a large scale. / As microalgas, ricas em lipídios, carboidratos e outros bioprodutos, têm sido consideradas como uma promissora fonte de matéria-prima graxa para a produção de biodiesel. Apesar do grande potencial e das vantagens normalmente apontadas para seu uso, ainda existem entraves científicos e tecnológicos que inviabilizam a produção de biodiesel de microalgas em larga escala. Desta forma, essa pesquisa teve como objetivo elaborar estudos técnico-científicos inovadores que possam contribuir para avaliar a viabilidade do uso das microalgas dulcícolas como fonte de matéria-prima graxa para a produção de biodiesel. Os trabalhos realizados permitiram avaliar a produção de biodiesel a partir da biomassa microalgal desde a seleção de cepas até a produção e análise da qualidade do biodiesel, e dessa forma possibilitou levantar parâmetros importantes sobre o cultivo e a produção do biodiesel de microalgas. Nesse trabalho de tese foi identificada uma microalga dulcícola com potencial para a produção de biodiesel - Choricystis minor var. minor – a qual apresentou teores e perfis de ácidos graxos adequados para a produção desse biocombustível, fornecendo 115% mais biodiesel que a semente de soja, via transesterificação direta da biomassa. Nos estudos sobre meios de cultivos observou-se que o meio WC é o melhor para a produção de biodiesel, em comparação ao meio BBM, sendo este último o mais adequado para a produção de pigmentos, tais como carotenoides e clorofila. A produção em escala piloto da microalga C. minor e a conversão da biomassa microalgal em biodiesel permitiu concluir que esta microalga apresentou produtividade volumétrica cerca de 11 vezes superior à soja, com a produção estimada em 6 toneladas de biodiesel por hectare ao ano. Alguns poucos ajustes são necessários para que todos os parâmetros de qualidade do biodiesel produzido possam alcançar a especificação adotada pela ANP. Dessa forma, conclui-se que a produção de biodiesel utilizando microalgas pode vir a ser uma realidade em grande escala.

Made available in DSpace on 2017-07-10T17:59:28Z (GMT). No. of bitstreams: 1 Sidney de O Novaes Junior.pdf: 6661390 bytes, checksum: db911a668b5af886529a0d861278a4c1 (MD5) Previous issue date: 2013-08-09 / This study aimed to assess the potential growth of two species of microalgae ( Chlorella vulgaris and Scenedesmus subspicatus ) in two types of effluents ( humans and pigs ) in order to obtain lipids that could be converted into biodiesel . Such compounds must meet the specifications contained in Resolution No. 07 / 2008 of ANP . The effluents were collected in human Station Wastewater Treatment III , located in the city of Foz do Iguaçu / PR and swine effluent in Granja São Jorge ( Colombari ) , located in the municipality of São Miguel do Iguaçu / PR . Both were sent to laboratories for analysis IAP and NUCLEOTEC constatassem that the presence of nitrogenous and phosphatic and oils and greases total , respectively. The effluents were diluted to previously autoclaved and which can serve as culture media for microorganisms . They were then inoculated samples of each type of microalgae in an effluent and evaluated their growth. In four vials was added the compound mineral Agrostemin ® to assess their influence on the results . Prepared growth curves after 60 days of cultivation samples were filtered under vacuum for further extraction of oils using ultrasound. The samples were sent for analysis of lipid content and composition in terms of fatty acids by gas chromatography , which demonstrated the presence of fatty acids that make up biodiesel . The physico-chemical characterization of the effluent showed the presence of small amounts of fats and greases in both the total effluent . The quantitative analysis of lipids showed the highest concentration in the cultivation of C. vulgaris in human effluent , with 50.12 % . With the addition of Agrostemin ®, the yield was better in the cultivation of S. subspicatus in swine wastewater , with 80% increase in the production of lipids , followed by the growth of C. vulgaris in human effluent , with 41 % . The acids were higher concentrations linoleic (C18 : 2) , the cultivation of C. vulgaris in human effluent with 35.17 % , followed by the arachidic ( 20:0 ) with the same microalgae , now in swine wastewater , with 37.29 % , the palmitoleic ( C16 : 1 ) arising from the cultivation of S. subspicatus in human effluent , with 28.85% and pentadecanoic ( C15 : 1 ), the culture of the same organism in swine wastewater , with 55.22 % . It was found that the latter is what gives the best results, but the viability of the remainder is also promising. / Este trabalho teve como objetivo avaliar o potencial de crescimento de duas espécies de microalgas (Chlorella vulgaris e Scenedesmus subspicatus) em dois tipos de efluentes (humanos e suínos) com a finalidade de se obter lipídios que pudessem ser convertidos em biodiesel. Tais compostos precisam atender às especificações contidas na Resolução nº 07/2008 da ANP. Os efluentes humanos foram coletados na Estação de Tratamento de Efluentes III, localizada na cidade de Foz do Iguaçu/PR e os efluentes suínos na Granja São Jorge (Colombari), localizada no município de São Miguel do Iguaçu/PR. Ambos foram enviados aos laboratórios do IAP e NUCLEOTEC para análises que constatassem a presença de componentes nitrogenados e fosfatados e de óleos e graxas totais, respectivamente. Os efluentes foram previamente autoclavados e diluídos para que pudessem servir de meios de cultura para os microrganismos. Foram, então, inoculadas amostras de cada microalga em um tipo de efluente e avaliado o seu crescimento. Em quatro frascos foi acrescentado o composto mineral Agrostemin® para avaliar sua influência nos resultados. Elaboradas as curvas de crescimento, após 60 dias de cultivo as amostras foram filtradas a vácuo para posterior extração dos óleos utilizando ultrassom. As amostras foram enviadas para análise do teor de lipídios e sua composição em termos de ácidos graxos por cromatografia gasosa, onde se constatou a presença de ácidos graxos que compõem o biodiesel. A caracterização físico-química dos efluentes constatou a presença de pequena quantidade de gorduras e graxas totais em ambos os efluentes. A análise quantitativa dos lipídios apontou a maior concentração no cultivo de C. vulgaris em efluentes humanos, com 50,12%. Com a adição do Agrostemin®, o melhor rendimento foi no cultivo de S. subspicatus em efluentes suínos, com aumento de 80% na produção de lipídios, seguido do cultivo de C. vulgaris em efluentes humanos, com 41%. Os ácidos com maiores concentrações foram o linoleico (C18:2), do cultivo de C. vulgaris em efluente humano com 35,17%, seguido do araquídico (20:0) com a mesma microalga, agora em efluente suíno, com 37,29%, o palmitoleico (C16:1) oriundo do cultivo de S. subspicatus em efluente humano, com 28,85% e o pentadecanoico (C15:1), da cultura do mesmo microrganismo em efluente suíno, com 55,22%. Constatou-se que este último é o que apresenta os melhores resultados, mas a viabilidade do restante é igualmente promissora.

Il s’agit de l’étude des successions écologiques du phytoplancton dans la lagune de Ghar El Melh au niveau de 5 stations pendant (i) un suivi bimensuel allant du janvier 2011 à janvier 2012 et (ii) un suivi saisonnier à partir du novembre 2012 à mars 2013. L’étude écologique a été consacrée au microphytoplancton en relation avec les facteurs abiotiques (température, salinité et nutriments) et biotiques (microalgues épiphytes, kystes des dinoflagellés, ultraplancton et ciliés). L’analyse hydrologique du milieu a montré une variation principalement saisonnière de la plupart des paramètres avec des valeurs élevées de la température et de la salinité en été et des concentrations exceptionnelles des nutriments en automne et en hiver. Le phytoplancton de la lagune de Ghar El Melh a été principalement dominé par les dinoflagellés et les diatomées avec une manifestation alarmante des espèces nuisibles atteignant plus que 70 % du peuplement. Ces proliférations ont été remarquées durant toute l’année avec une manifestation plus marquante dans les régions internes de la lagune ce qui est principalement tributaire à la température de l’eau, l’azote total, le nitrogène et l’orthophosphate. Une importante contribution des espèces épiphytes au phytoplancton a été signalée notamment celle du dinoflagellé toxique Prorocentrum lima. L’inventaire spécifique du phytoplancton dominé par l’espèce Prorocentrum micans (> 28 %) et d’autres espèces ne produisant pas de kyste a été en concordance avec les faibles valeurs enregistrées pour les kystes de dinoflagellés dans le sédiment où le maximum enregistré a été de l’ordre de 229 kystes g-1. Une part importante de l’ultraplancton a été représentée par le nanophytoplancton et les hétérotrophes procaryotes, dépassant largement les concentrations microphytoplanctoniques, ce qui est un indice de l’eutrophisation du milieu. Des corrélations hautement significatives ont été trouvées entre le microphytoplancton et les organismes microzooplanctoniques évoquant un probable rôle de ces derniers dans le contrôle des proliférations microalguales nuisibles. / We explored the distribution of the microphytoplankton community in relation to both abiotic (temperature, salinity and nutrients) and biotic factors (epiphytes microalgae, dinoflagellate cysts, ultraplankton and ciliates) in Ghar al Melh Lagoon at 5 stations during (i) a biweekly sampling from January 2011 to January 2012 and (ii) a seasonal sampling from november 2012 to march 2013. The results pointed out a seasonal variation of all parameters with high water temperature and salinity in summer and exceptional concentrations of nutrients in autumn and winter. Microphytoplankton was mainly dominated by dinoflagellates and diatoms with sequences of harmful species proliferation attaining more than 70 % of total phytoplankton. These sporadic proliferations occurred throughout the year with exacerbation in the inner parts of the lagoon mainly related to temperature and total nitrogen, nitrogen and orthophosphate. An important contribution of epiphytic species to phytoplankton abundance was recorded with especially the presence of the toxic dinoflagellate Prorocentrum lima. Microphytoplankton was dominated by Prorocentrum micans (> 28 %) and other non-cyst former species consistent with the low dinoflagellate cysts found at sediment surface (maximum = 229 cysts g-1 DS). Urtraplankton was chiefly represented by nanophytoplankton and heterotrophic prokaryotes, with low microphytoplanktonic abundance. This indicates that the lagoon is undergoing eutrophication. Significant correlations were reported between phytoplankton and microzooplankton suggesting a potential role of the former in controlling harmful microalgae proliferations.

Microalgae are recognized sources of metabolites with high added value, largely by biological activity of these compounds. Several species of cyanobacteria produce secondary metabolites with bioactive character due to its molecular structure. Currently studying a lot about pigments for its biological value, giving emphasis on its bioactivity, but chlorophyll is still poorly understood. Despite its important biological role, research on chlorophyll and its potential antioxidant and pro-oxidant are still scarce. The lack of information can be attributed to the difficulty of obtaining chlorophyll and its derivatives in a purified form and also to their instability, which triggers a process of degradation. Many uncertainties still exist in the scientific field of the natural chlorophyll, their metabolites, impact on health, due to its high content in some vegetables, widespread occurrence in nature, and its degradation during food processing and human digestion. The production of compounds of chlorophyll by microalgae, due to their diverse metabolism, makes biomass microalgal an alternative source, with different chemical structures as compared to conventional food matrix, and its complex system of conjugated bonds on pyrrole rings enables an antioxidant character, or pro-oxidative, acting in scavenging free radicals or reactive triggering systems of molecular degradation. In this sense, the study of these chlorophyll pigments designed to further focus on their chemical structure may help to elucidate the real potential bioactive these substances. Given the various chlorophyllous structures present in Phormidium autumnale extract with different polarities, it is crucial to choose the appropriate method for evaluating the antioxidant potential, and to investigate the antioxidant mechanism developed by each structure of the molecule. A total of eleven chlorophyll compounds were separated on biomass Phormidium autumnale the photosynthetic culture. Among the major compounds found chlorophyll a (1498.40μg / g) and pheophytin b (613.23μg / g), where the total chlorophyll pigments totaled 2720.19μg / g in the biomass extract. The potential for scavenging free radicals was examined by two different methods with regard to their polarity, being that for the lipophilic method of the Phormidium extract autumnale proved to be 200 times more potent than α-tocopherol at the disposal of peroxyl radicals , and the method the hydrophilic Phormidium autumnale extract showed antioxidant activity of 219.82 mu equivalent Trolox / g extract. Finally, we note that the biomass Phormidium autumnale has considerable potential in the production of chlorophyll compounds in phototrophic conditions, proving to be an alternative source to conventional arrays of chlorophyll pigments and proved efficient in eliminating free radicals, showing their potential bioactive . / Microalgas são fontes reconhecidas de metabólitos com alto valor agregado, em grande parte pela atividade biológica destes compostos. Diversas espécies de cianobactérias produzem metabólitos secundários com caráter bioativo em função de sua estrutura molecular. Atualmente estuda-se muito a respeito de pigmentos pelo seu valor biológico, dando-se ênfase em sua bioatividade, porém a clorofila ainda é pouco elucidada. Apesar de seu importante papel biológico, as pesquisas sobre clorofila e seu potencial antioxidante e pró-oxidante ainda são escassas. A escassez de informações pode ser atribuída à dificuldade de se obter clorofilas e seus derivados em uma forma purificada e também à sua instabilidade, o que desencadeia um processo de degradação. Muitas incertezas ainda existem no campo científico sobre a clorofila natural, seus metabólitos, impacto sobre a saúde, devido seu alto conteúdo em alguns vegetais, ocorrência generalizada na natureza, e sua degradação durante o processamento de alimentos e digestão humana. A produção de compostos clorofilados pelas microalgas, em função de seu metabolismo diversificado, torna a biomassa microalgal uma fonte alternativa, com estruturas químicas diferenciadas quando comparada às matrizes alimentícias convencionais, sendo que seu sistema complexo de ligações conjugadas em anéis pirrólicos possibilita um caráter antioxidante, ou pró-oxidativo, atuando no sequestro de radicais livres ou desencadeando sistemas reacionais de degradação molecular. Neste sentido, o estudo destes pigmentos clorofilados visando um maior enfoque em suas estruturas químicas poderá auxiliar na elucidação do real potencial bioativo destas substâncias. Diante das diversas estruturas clorofiladas presentes no extrato de Phormidium autumnale, com diferentes polaridades, é fundamental escolher o método adequado para a avaliação do potencial antioxidante, além de investigar o mecanismo antioxidante desenvolvido por cada estrutura da molécula. Um total de onze compostos clorofilados foram separados na biomassa de Phormidium autumnale no cultivo fotossintético. Entre os compostos majoritários foram encontrados clorofila a (1498.40μg / g), e feofitina b (613.23μg / g), onde os pigmentos totais clorofilados totalizaram 2720.19μg / g no extrato de biomassa. O potencial de eliminação de radicais livres foi analisado por dois métodos diferentes no que diz respeito à sua polaridade, sendo que para o método lipofílico o extrato de Phormidium autumnale mostrou-se 200 vezes mais potente do que o α-tocoferol na eliminação de radicais peroxil, e para o método hidrofílico o extrato de Phormidium autumnale apresentou atividade antioxidante de 219,82 mμ Trolox equivalente / g de extrato. Finalmente, observamos que a biomassa de Phormidium autumnale apresenta um potencial considerável na produção de compostos clorofilados em condições fototróficas, provando ser uma fonte alternativa para matrizes convencionais de pigmentos de clorofila, bem como se mostrou eficiente na eliminação de radicais livres, evidenciando seu potencial bioativo.

Microalgae have a variety of commercial applications, the oldest of which include utilisation as a food source and for use in wastewater treatment. These applications, however, are seldom combined due to toxicity concerns, for ethical reasons, and generally the requirement for cultivation of a single algae species for use as a feed supplement. These problems might be negated if a “safer” wastewater such as that from agricultural and/or commercial food production facilities were to be utilised and if a stable algae population can be maintained. In this investigation preliminary studies were carried out using an Integrated Algae Pond System (IAPS) for domestic wastewater treatment to determine the species composition in the associated High Rate Algae Ponds (HRAPs). The effect of different modes of operation, continuous versus batch, on nutrient removal, productivity and species composition was also investigated. Furthermore, indigenous species in the HRAP were isolated and molecularly identified as, Chlorella, Micractinium, Scenedesmus and Pediastrum. Additionally, the effect of the nor amino acid, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) and its Cu-chelated derivative, on the growth and biochemical composition of Chlorella, Micractinium, Scenedesmus, Pediastrum and Spirulina was investigated. Species composition in the HRAP was stable under continuous operation with Micractinium dominating > 90% of the algae population. Under batch operation the population dynamic shifted; Chlorella outcompeted Micractinium possibly due to nutrient depletion and selective grazing pressures caused by proliferation of Daphnia. Higher species diversity was observed during batch mode as slower growing algae were able to establish in the HRAP. Nutrient removal efficiency and biomass productivity was higher in continuous mode, however lower nutrient levels were obtained in batch operation. HMTBA did not significantly affect growth rate, however treatment with 10 mg.L-1 resulted in slightly increased growth rate in Micractinium and increased final biomass concentrations in Chlorella, Micractinium and Spirulina (although this was not statistically significant for Micractinium and Spirulina), which are known mixotrophic species. Algae treated with Cu-HMTBA, showed reduced final biomass concentration with 10 mg.L-1, caused by Cu toxicity. Biochemical composition of the algae was species-specific and differed through the growth cycle, with high protein observed during early growth and high carbohydrate during late growth/early stationary phase. Additionally, 0.1 mg.L-1 HMTBA and Cu-HMTBA significantly reduced protein content in Chlorella, Micractinium, Scenedesmus and Pediastrum. In conclusion, operation of the HRAP in continuous culture provided suitable wastewater treatment with high productivity of an ideal species, Micractinium, for use in animal feed supplementation. This species had 40% protein content during growth (higher than the other species tested) and dominated the HRAP at > 90% of the algae population during continuous mode. Addition of HMTBA (> 1 mg.L-1) to algae cultivation systems and those treating wastewater, has the potential to improve productivity and the value of the biomass by enhancing protein content. Overall, the co-utilisation of microalgae for wastewater treatment and the generation of a biomass rich in protein, for incorporation into formulated animal feed supplements, represents a closed ecosystem which conserves nutrients and regenerates a most valuable resource, water.

Algae have been the centre of recent research as a sustainable feedstock for fuel because of their higher oil yield in comparison to other plant sources. However, algae biofuel still performs poorly from an economic and environmental perspective due to the high reliance on freshwater and nutrients for cultivation, among other challenges. The use of wastewater has been suggested as a sustainable way of overcoming these challenges because wastewater can provide a source of water and nutrients for the algae. Moreover, the ability of the algae to remove contaminants from wastewater also enhances the total economic output from the cultivation. However, the success of this strategy still depends greatly on efficient strain selection, cultivation and harvesting. Therefore, this PhD thesis has focussed on strain isolation, characterisation, optimisation and cultivation in open pond systems. Five algae strains were isolated from wastewater treatment tanks at a municipal water treatment plant in North West England. The isolated strains were morphologically and genetically characterised as green single-celled microalgae: Chlamydomonas debaryana, Hindakia tetrachotoma, Chlorella luteoviridis, Parachlorella hussii and Desmodesmus subspicatus. An initial screening of these strains concluded that C. luteoviridis and P. hussii were outstanding in all comparisons and better than some of the strains previously reported in the literature. Further tests carried out to elucidate the underlying tolerance mechanisms possessed by these strains were based on stress tolerance and acclimation hypotheses. In the following experiments, C. luteoviridis and P. hussii were found to have higher anti-oxidant enzyme activity that helps in scavenging reactive oxygen species produced as a result of exposure to wastewater. This result provides a new argument for screening microalgae strains for wastewater cultivation on the basis of anti-oxidant activity. In addition, the two strains could grow heterotrophically and are better adapted to nutrient deficiency stress than the other three isolates. In order to understand the role of microalgae acclimation in wastewater cultivation, strains identical or equivalent to the wastewater treatment tank isolates were obtained from an algae culture collection. These strains had not been previously exposed to wastewater secondary effluent. The initial growth of these strains in wastewater secondary effluent was very poor. However, after two months of acclimation to increasing concentrations of secondary wastewater effluent, it was observed that growth, biomass and lipid productivities of most of the strains were significantly improved, although still not as high as the indigenous strains. Therefore, it was concluded that continuous acclimation is an additional factor to the successful growth of algae in wastewater. Furthermore, addition of 25% activated sludge centrate liquor to the secondary effluent was found to increase algal growth and biomass productivity significantly. Futher tests to examine the continous cultivation of C. luteoviridis and P. hussii in wastewater showed that a biomas productivity of 1.78 and 1.83 g L-1 d-1 can be achieved on a continual basis. Finally, the capability of C. luteoviridis and P. hussii for full seasonal cultivation in a 150 L open pond in a temperate climate was studied, using the optimised secondary wastewater +25% liquor medium. Each strain was capable of growth all year including in autumn and winter but with strongest growth, productivity and remediation characteristics in the summer and spring. They could maintain monoculture growth with no significant contamination or culture crash, demonstrating the robustness of these strains for wastewater cultivation in a northern European climate.

Les lipides des microalgues et les glucides de cyanobactéries peuvent être transformés en biodiesel et en bioéthanol, respectivement. L'amélioration de la production de ces molécules doit prendre en compte les entrées périodiques (principalement la lumière) forçant le réseau métabolique de ces organismes photosynthétiques. Il est donc nécessaire de tenir compte de la dynamique du réseau métabolique en réduisant sa dimension pour assurer la maniabilité mathématique. Le but de ce travail est de concevoir une approche originale pour réduire les réseaux métaboliques dynamiques tout en conservant la dynamique de base. Cette méthode est basée sur une séparation en échelles de temps. Pour une classe de modèles de réseaux métaboliques décrits par des ODE, la dynamique des systèmes réduits est calculée à l'aide du théorème de Tikhonov pour les systèmes singulièrement perturbés. Cette approximation quasi-stationnaire coïncide avec la dynamique du réseau d'origine, avec une erreur bornée. L'approche est d'abord développée pour les systèmes de réaction pouvant être linéarisés autour d'un point de travail et forcés par des entrées continues. Ensuite, une généralisation de cette méthode est donnée pour les réseaux à réactions rapides de cinétiques de Michaelis-Menten et tout type de cinétiques lentes, prenant également en compte un nombre fini d'entrées continues externes. La méthode de réduction met en évidence une relation entre la grandeur de la concentration des métabolites et la gamme des vitesses de réaction : les métabolites consommés par les réactions rapides ont une concentration inférieure d'un ordre de grandeur à celle des métabolites consommés à faible vitesse. Cette propriété est satisfaite pour les métabolites à dynamique rapide ne se trouvant pas dans un piège de flux, concept introduit dans ce travail. Le système réduit peut être calibré avec des données expérimentales à l'aide d'une procédure d'identification dédiée basée sur la minimisation. L'approche est illustrée par un réseau métabolique de microalgues autotrophes, comprenant le métabolisme central et représentant la dynamique des glucides et des lipides. Cette approche permet de bien ajuster les données expérimentales de Lacour et al. (2012) avec la microalgue Tisochrysis lutea. Enfin, un schéma visant à optimiser la production de molécules cibles est proposé en utilisant le système réduit. / Lipids from microalgae and carbohydrates from cyanobacteria can be transformed into biodiesel and bioethanol, respectively. Enhancing the production of these molecules must account for the periodic inputs (mainly light) forcing the metabolic network of these photosynthetic organisms. It is therefore necessary to account for the dynamics of the metabolic network, while reducing its dimension to ensure mathematical tractability. The aim of this work is to design an original approach to reduce dynamic metabolic networks while keeping the core dynamics. This method is based on time-scale separation. For a class of metabolic network models described by ODE, the dynamics of the reduced systems are computed using the theorem of Tikhonov for singularly perturbed systems. This Quasi Steady State Approximation accurately coincides with the original network dynamics, with a bounded error. The approach is first developed for reaction systems that can be linearized around a working point and that are forced by external continuous inputs. Then, a generalization of this method is given for networks with fast reactions of Michaelis-Menten kinetics and any type of slow kinetics, also considering a finite number of external continuous inputs. The reduction method highlights a relation between the concentration magnitude of the metabolites and the range of the reaction rates: the metabolites that are consumed by fast reactions have concentration one order of magnitude lower than metabolites consumed at slow rates. This property is satisfied for metabolites with fast dynamics that are not in a flux trap, a concept introduced in this work. The reduced system can be calibrated with experimental data using a dedicated identification procedure based on minimization. The approach is illustrated with an autotrophic microalgae metabolic network, including the core metabolism and representing the carbohydrates and lipids dynamics. The approach efficiently fits the experimental data from Lacour et al. (2012) with the microalgae Tisochrysis lutea. Finally, a scheme to optimize the production of target molecules is proposed using the reduced system.

La protection intégrée, qui vise à réduire l’usage des pesticides, est un défi majeur pour l’agriculture du XXIème siècle. Le développement de nouvelles approches agronomiques qui concilient environnement et agriculture est une condition indispensable pour l’agriculture de demain. Dans ce contexte, l’utilisation d’éliciteurs capables de mimer une attaque pathogène et de promouvoir un état de résistance chez les plantes face à des maladies représente une alternative naturelle à la lutte chimique. Ces éliciteurs sont nommés les stimulateurs de défense des plantes (SDP). Ils peuvent provenir de différentes sources et être extraits à partir de macroalgues comme c’est le cas des SDP à base de polysaccharides d’algues tels que la laminarine utilisée pour stimuler l’immunité de plantes agronomiques. Toutefois, l’exploitation de ces ressources dans leur milieu naturel et les difficultés de production liées à leur cycle de développement constituent des freins à leur utilisation. La valorisation des microalgues comme source de SDP pourrait permettre de s’affranchir de ces contraintes. Cependant la recherche et de molécules SDP chez les microalgues est encore peu abordée. Au cours de ce travail, le potentiel d’une culture de microalgue, Phaeodactylum tricornutum, à induire des réactions de défense chez les plantes a été évalué. Un broyat cellulaire a été appliqué sur des plantules d’Arabidopsis thaliana. Le caractère éliciteur de ce broyat a été testé et caractérisé par des approches microscopiques, physiologiques et moléculaires. Les résultats ont montré que les plantes traitées présentaient des niveaux d’expression des gènes PR-1, PAD3, ACS6 et WRKY40 et un niveau de protection contre la bactérie Pseudomonas syringae DC3000 (Pst) plus élevés que les plantes non traitées. De plus, un effet bactéricide in vitro sur la bactérie Pst a été observé. Ces résultats offrent de nouvelles perspectives pour le développement de produits SDP d’origine naturelle capables de protéger les cultures. / Integrated plant protection, which aims to reduce the use of pesticide, is a major challenge for the agriculture of the 21st century. The development and application of new agronomic approaches is a prerequisite for crop protection in a sustainable agriculture system. In this context, the use of elicitors capable of mimicking a pathogenic attack and promoting a plant resistance state against diseases is a natural alternative to the use of agro-chemicals. These elicitors are also called plant defense stimulators (PDS). These can be obtained from different sources including macroalgae as it the case for the polysaccharide-based PDS laminarin that is currently used for the protection of a number of crops. However, the exploitation of these natural resources and the difficulties of their production due to their development cycle do hamper their use at a large scale. One of the possibilities to overcome these difficulties is the use of microalgae as a source of PDS. But this possibility and the potential of microalgaederived PDS for crop protection are currently under investigated. In the present work, we have used a cell extract from the microalgae Phaeodactylum tricornutum and assessed its defense response-eliciting activities on Arabidopsis thaliana seedlings by using microscopic, physiological and molecular approaches. The results show that treated plants exhibit higher levels of expression of the PR-1, PAD3, ACS6 and WRKY40 genes and a higher level of protection against the pathogenic bacterium Pseudomonas syringae DC3000 (Pst) than nontreated plants. An In vitro antibacterial activity on the Pst bacteria was also observed. Our findings suggest that P. tricornutum cell extracts are able to activate plant immune responses and offer new perspectives for the development of novel plant defense stimulators.

Karotenoidy jsou přírodní pigmenty vyskytující se v mikroorganismech jako jsou řasy, kvasinky a sinice. Představují nejrozšířenější skupinu antioxidantů s významným biologickým účinkem. V současnosti vzrůstá zájem o karotenoidy vzhledem k jejich příznivým vlivům na lidské zdraví. Chlorofyly jsou zelená fotosyntetická barviva, která nacházejí uplatnění v potravinářství jako intenzivní zelená barviva. Koenzym Q je znám svým pozitivním vlivem pro správnou funkci řady orgánů v lidském těle. Ergosterol je nedílnou součástí membrán kvasinek a hub. Je to provitamin D2, který je důležitou součástí imunitního systému. Mikrobiální lipidy, nebo také ‚‚Single cell oils‘‘ jsou charakteristické vysokým obsahem zdraví prospěšných nenasycených mastných kyselin, které lze využít ve farmacii či kosmetice. Mikrobiální lipidy jsou dále studovány jako alternativa pro výrobu biopaliv. Dizertační práce byla zaměřena na studium a možnosti optimalizace produkce lipidů a lipidických látek vybranými kmeny karotenogenních kvasinek, mikrořas a sinic. V rámci práce byly testované kvasinky rodu Rhodotorula, Rhodosporidium, Cystofilobasidium a Sporidiobolus podrobené kultivacím na sérií médií s různými C/N poměry v rozsahu 13 až 100, obsahujících upravené odpadní substráty z potravinářského průmyslu. Vybrané kmeny byly poté kultivovány v bioreaktorech v médiu obsahujícím kombinaci odpadních substrátů. Kultivace mikrořas rodu Desmodesmus, Scenedesmus, Chlorella, Coccomyxa, Chlamydomonas, Botryococcus se zabývaly optimalizací jednotlivých komponent média a aplikací různých stresů s cílem navýšení produkce studovaných metabolitů. V rámci experimentů s extrémofilní mikrořasou Coccomyxa byly provedeny pilotní velkoobjemové kultivace v otevřených nádržích. V závěrečné části byl provedeny pilotní screeningové a velkoobjemové bioreaktorové experimenty zaměřené na možnosti kokultivace karotenogenních kvasinek a mikrořas. Testované kmeny kvasinek byly s rozdílnou úspěšností schopny utilizovat média obsahující hydrolyzované odpadní substráty. Nejlepším kmenem byl Sporidiobolus pararoseus, který na médiích dosahoval nejvyšších produkcí biomasy i sledovaných metabolitů. Z testovaných odpadních substrátů byla nejlepší kombinace odpadního fritovacího oleje a hydrolyzátu kávové sedliny. Úspěšná optimalizace složení hlavních komponent minerálního média vedla k zvýšené produkci studovaných metabolitů. Největší vliv měl optimální poměr P/N a aplikace oxidačního stresu. Nejlepších výsledků dosáhly mikrořasy rodu Desmodesmus a Scenedesmus. Velkoobjemové kultivace Coccomyxy onubensis potvrdily rezistenci kultury proti kontaminaci vnějšími vlivy a schopnost růstu za vysoké teploty a intenzity světelného záření. Kokultivační experimenty potvrdily schopnost symbiotického růstu kvasinek a mikrořas. Nejlepších výsledků dosahovaly všechny testované kvasinky s mikrořasami rodu Demsodesmus a Scenedesmus a v menší míře i rodu Coccomyxa.

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