Il n’est pas constaté d’autofloculation naturelle dans les cultures hypersalines de D. salina. De plus, cette dernière ne flotte pas sans déstabilisation de la suspension. La floculation induite par augmentation du pH lors de l’ajout de soude a été choisie dans cette étude pour permettre la flottation. Des efficacités de récupération supérieures à 90% et des facteurs de concentration autour de 20 sont atteints. Un mécanisme d’autofloculation, avec précipitation de Mg(OH)2 est proposé pour expliquer la floculation par balayage de D. salina. L’influence du débit d’injection de soude ajoutée est aussi étudié pour anticiper les contraintes relatives à l’industrialisation du procédé. Ce débit n’a pas d’effet sur l’efficacité de récupération des algues et réduit le facteur de concentration seulement pour les injections abruptes.L’augmentation naturelle du pH de culture par l’intermédiaire de l’activité photosynthétique pourrait permettre de réduire le taux de base consommé. Les cellules non-récoltées restent en vie durant l’augmentation du pH et pourraient être utilisées comme inoculum d’une nouvelle culture. / Natural autoflocculation was not observed in a Dunaliella salina hypersaline culture and the microalgae did not float without destabilization of the algal suspension. High-pH-induced flocculation by sodium hydroxide addition was chosen to induce flotation. Recovery efficiencies greater than 90% and concentration factors of around 20 were reached. An autoflocculation mechanism, with precipitation of magnesium hydroxide, is proposed to explain a sweeping flotation of D. salina cells. The influence of the flow rate of sodium hydroxide addition was also studied to anticipate the constraints related to the industrialization of this process. The flow rate of sodium hydroxide addition had no effect on the recovery efficiency and reduced the concentration factor only for abrupt injections. Natural increase of culture pH by photosynthetic activity could reduce the amount of base consumed. Non-harvested cells remained viable during pH increase and could be used as inoculum for a new culture.

Les microalgues constituent une source prometteuse de biocarburants dits de troisième génération. L'intérêt de ces micro-organismes photosynthétiques réside également dans l'étendue de la palette de molécules qu'elles peuvent produire, telles que les protéines, les pigments ou encore les vitamines. Néanmoins, des progrès sont encore nécessaires pour diminuer les coûts économiques et environnementaux des procédés de culture et assurer ainsi la viabilité de la filière. En particulier, mieux comprendre l'effet de la lumière sur la productivité des cultures denses est une étape essentielle pour optimiser ces procédés. L'objectif de cette thèse est d'étudier les effets quantitatifs et qualitatifs de la lumière sur la croissance et les mécanismes d'acclimatation de deux espèces de microalgues à fort intérêt biotechnologique, Dunaliella salina et Tisochrysis lutea. La première partie de cette thèse examine la réponse de Dunaliella salina à des variations rapides de la lumière, reproduisant les fluctuations de l'éclairement typiquement perçues par les cellules microalgales brassées au sein des systèmes de cultures industriels à haute densité de type raceway. Dans la seconde partie, nous avons analysé la réponse de Dunaliella salina et Tisochrysis lutea à différentes compositions du spectre lumineux. L'approche à la fois expérimentale et théorique nous a permis d'identifier les effets d'une lumière colorée sur la productivité et la composition pigmentaire des microalgues. Nos résultats offrent des perspectives encourageantes et des pistes concrètes permettant d'optimiser l'utilisation de la lumière pour produire des microalgues et d'améliorer le bilan énergétique de ces procédés. / Microalgae are a promising source of third generation biofuels. The interest on these photosynthetic microorganisms also lies within the extent of the spectrum of molecules that they can produce, such as proteins, pigments, and vitamins. Nevertheless, further progress is still necessary to reduce the economic and environmental costs of cultivation processes and to ensure the viability of this sector. In particular, better understanding of the effect of light on the productivity of dense cultures is an essential step to optimize these processes. The aim of this thesis is to study the quantitative and qualitative effects of light on growth and acclimation mechanisms of two species of microalgae with high biotechnological interest ; Dunaliella salina and Tisochrysis lutea. The first part of this thesis examines the response of Dunaliella salina to rapid light changes, by reproducing irradiance fluctuations typically experienced by microalgal cells in a highly turbid suspension in raceway ponds. In the second part, we analyzed the response of Dunaliella salina and Tisochrysis lutea to different light spectra. Our experimental and theoretical approach allowed us to identify the effect of colored light on productivity and pigment composition of microalgae. Furthermore, our results offer encouraging prospects for elevate understanding and use of light and improve the energy performance of these processes.

O presente trabalho avaliou a influência de quatro fatores no processo de extração dos lipídios da biomassa de três microalgas marinhas: Chlorella minutíssima, Dunaliella salina e Nannochloropsis gaditana. Estes fatores foram: a umidade da biomassa, a proporção entre os solventes, o volume total de solventes e o tempo de ultrassom, estabelecidos em três diferentes níveis e verificados simultaneamente em diferentes combinações, em um arranjo ortogonal de Taguchi. O objetivo primeiro deste estudo simultâneo foi encontrar o ajuste fornecedor da maior porcentagem de lipídios. Três diferentes níveis de umidade foram investigados na biomassa: 64%, 72% e 80%. A extração de lipídios da biomassa microalgal com elevado teor de umidade permite grande economia de tempo e energia, que seriam consumidos no processo de secagem, caso o processo de extração exigisse somente biomassa seca. Quanto à proporção entre os solventes e ao volume total de solventes, buscou-se encontrar as quantidades ideais de cada um dos solventes empregados: clorofórmio, metanol e água, que contribuem para a lise celular e o sequestro mais eficiente dos lipídios. A quarta variável, o tempo de ultrassom, foi verificado por ser um catalisador do processo de lise celular. O estudo dessas variáveis visou não somente melhorar a eficácia da extração, mas economizar tempo de processo e energia. O processo compreendeu a hidratação controlada da biomassa, seguido da extração propriamente dita, onde um sistema ternário formou-se com a separação posterior em duas fases distintas, com os lipídios dissolvidos na fase orgânica. Além de determinar a melhor condição de extração, este trabalho verificou o potencial do material lipídico das três espécies à conversão em ésteres de etila, visando a produção de biodiesel e a composição em ácidos graxos, avaliando seu potencial à aplicação nutracêutica, como fonte de ácidos graxos essenciais. A melhor condição de extração resultou de uma biomassa com 64% de umidade, proporção de 5,7 de clorofórmio para 3,0 de metanol e 1,0 de água em um volume total de 33 mL de solventes por grama de biomassa; tempos de ultrassom de 50 ou 70 minutos mostraram-se adequados para obtenção dos maiores teores lipídicos. O material lipídico extraído das três espécies revelou-se promissor como matéria-prima para a produção de biodiesel, com conversões de até 94,5%, via catálise ácida, após um tempo de reação de 5 horas a 80ºC e também rico em ácidos graxos essenciais, principalmente os ácidos linoleico e ?-linolênico. A composição em ácidos graxos das três espécies foi comparada com outras espécies do mesmo gênero, e diferentes meios de cultivo, mostrando a influência do cultivo sem injeção de dióxido de carbono, que favorece a síntese de ácidos graxos poli-insaturados. O método de Taguchi foi o diferencial por permitir a avaliação conjunta de todas as variáveis, contribuindo para encontrar os melhores ajustes em menor tempo, ressaltando o ineditismo deste trabalho de pesquisa. / This work aimed to study four factors that influence in the process of lipids extraction in the biomass from three marine microalgae species: Chlorella minutíssima, Dunaliella salina and Nannochloropsis gaditana. These factors were: biomass\' moisture, solvents\' ratio, solvents\' total volume and ultrasonic time, which were set at three different levels and observed simultaneously in a Taguchi orthogonal arrangement. The first objective of this study was to find the simultaneous adjustment provider of higher percentage of lipids. Three different humidity levels were investigated in biomass: 64%, 72% and 80%. The extraction of lipids of microalgal biomass with high moisture content allows great savings of time and energy that would be consumed in the drying process before the extraction if only dry biomass was demanded. Regarding the proportion between solvents and the total volume of solvents, we looked for to find the optimal amounts of each of the employed solvent: chloroform, methanol and water, which contribute to cell lysis and more efficient lipids sequestration. The fourth variable as a catalyst cellular lysis process, ultrasound time was checked. The method comprised controlled hydration of the biomass followed by extraction, where a ternary system formed with subsequent separation into two phases, with lipids dissolved in the organic phase. Besides determining the best extraction condition, this work had as objective verifying the potential of the lipid material to conversion into ethyl esters, aiming at the production of biodiesel and the composition of fatty acids, evaluating its potential for nutraceutical application as a source of essential fatty acids. The best extraction condition employed 64% humidity biomass, ratio of 5,7/3/1 chloroform/methanol/water, 33 mL solvents/gram, ultrasound times of 50 or 70 minutes were adequate to obtain the highest lipid contents. The lipid material extracted from the three species proved promising as raw material for biodiesel production, with conversions up to 94.5%, via acid catalysis, after a reaction time of 5 hours at 80ºC and also rich in essential fatty acids, especially linoleic and ?-linolenic acids. The fatty acid composition of the three species was compared to other species of the same genus and different culture media, showing the influence of the culture without injection of carbon dioxide, which favors the synthesis of polyunsaturated fatty acids. Taguchi method was the differential for allowing the joint evaluation of all the variables, contributing to find the best adjustments in a shorter time, highlighting the novelty of this research work.

O presente trabalho avaliou a influência de quatro fatores no processo de extração dos lipídios da biomassa de três microalgas marinhas: Chlorella minutíssima, Dunaliella salina e Nannochloropsis gaditana. Estes fatores foram: a umidade da biomassa, a proporção entre os solventes, o volume total de solventes e o tempo de ultrassom, estabelecidos em três diferentes níveis e verificados simultaneamente em diferentes combinações, em um arranjo ortogonal de Taguchi. O objetivo primeiro deste estudo simultâneo foi encontrar o ajuste fornecedor da maior porcentagem de lipídios. Três diferentes níveis de umidade foram investigados na biomassa: 64%, 72% e 80%. A extração de lipídios da biomassa microalgal com elevado teor de umidade permite grande economia de tempo e energia, que seriam consumidos no processo de secagem, caso o processo de extração exigisse somente biomassa seca. Quanto à proporção entre os solventes e ao volume total de solventes, buscou-se encontrar as quantidades ideais de cada um dos solventes empregados: clorofórmio, metanol e água, que contribuem para a lise celular e o sequestro mais eficiente dos lipídios. A quarta variável, o tempo de ultrassom, foi verificado por ser um catalisador do processo de lise celular. O estudo dessas variáveis visou não somente melhorar a eficácia da extração, mas economizar tempo de processo e energia. O processo compreendeu a hidratação controlada da biomassa, seguido da extração propriamente dita, onde um sistema ternário formou-se com a separação posterior em duas fases distintas, com os lipídios dissolvidos na fase orgânica. Além de determinar a melhor condição de extração, este trabalho verificou o potencial do material lipídico das três espécies à conversão em ésteres de etila, visando a produção de biodiesel e a composição em ácidos graxos, avaliando seu potencial à aplicação nutracêutica, como fonte de ácidos graxos essenciais. A melhor condição de extração resultou de uma biomassa com 64% de umidade, proporção de 5,7 de clorofórmio para 3,0 de metanol e 1,0 de água em um volume total de 33 mL de solventes por grama de biomassa; tempos de ultrassom de 50 ou 70 minutos mostraram-se adequados para obtenção dos maiores teores lipídicos. O material lipídico extraído das três espécies revelou-se promissor como matéria-prima para a produção de biodiesel, com conversões de até 94,5%, via catálise ácida, após um tempo de reação de 5 horas a 80ºC e também rico em ácidos graxos essenciais, principalmente os ácidos linoleico e ?-linolênico. A composição em ácidos graxos das três espécies foi comparada com outras espécies do mesmo gênero, e diferentes meios de cultivo, mostrando a influência do cultivo sem injeção de dióxido de carbono, que favorece a síntese de ácidos graxos poli-insaturados. O método de Taguchi foi o diferencial por permitir a avaliação conjunta de todas as variáveis, contribuindo para encontrar os melhores ajustes em menor tempo, ressaltando o ineditismo deste trabalho de pesquisa. / This work aimed to study four factors that influence in the process of lipids extraction in the biomass from three marine microalgae species: Chlorella minutíssima, Dunaliella salina and Nannochloropsis gaditana. These factors were: biomass\' moisture, solvents\' ratio, solvents\' total volume and ultrasonic time, which were set at three different levels and observed simultaneously in a Taguchi orthogonal arrangement. The first objective of this study was to find the simultaneous adjustment provider of higher percentage of lipids. Three different humidity levels were investigated in biomass: 64%, 72% and 80%. The extraction of lipids of microalgal biomass with high moisture content allows great savings of time and energy that would be consumed in the drying process before the extraction if only dry biomass was demanded. Regarding the proportion between solvents and the total volume of solvents, we looked for to find the optimal amounts of each of the employed solvent: chloroform, methanol and water, which contribute to cell lysis and more efficient lipids sequestration. The fourth variable as a catalyst cellular lysis process, ultrasound time was checked. The method comprised controlled hydration of the biomass followed by extraction, where a ternary system formed with subsequent separation into two phases, with lipids dissolved in the organic phase. Besides determining the best extraction condition, this work had as objective verifying the potential of the lipid material to conversion into ethyl esters, aiming at the production of biodiesel and the composition of fatty acids, evaluating its potential for nutraceutical application as a source of essential fatty acids. The best extraction condition employed 64% humidity biomass, ratio of 5,7/3/1 chloroform/methanol/water, 33 mL solvents/gram, ultrasound times of 50 or 70 minutes were adequate to obtain the highest lipid contents. The lipid material extracted from the three species proved promising as raw material for biodiesel production, with conversions up to 94.5%, via acid catalysis, after a reaction time of 5 hours at 80ºC and also rich in essential fatty acids, especially linoleic and ?-linolenic acids. The fatty acid composition of the three species was compared to other species of the same genus and different culture media, showing the influence of the culture without injection of carbon dioxide, which favors the synthesis of polyunsaturated fatty acids. Taguchi method was the differential for allowing the joint evaluation of all the variables, contributing to find the best adjustments in a shorter time, highlighting the novelty of this research work.

Exploité depuis toujours, le sel a connu, au temps de la Gabelle notamment, des heures de gloire certaines, faisant de lui un métal blanc précieux, recherché et coûteux. Cependant, de nos jours, il est considéré comme un minéral essentiel, certes, mais aussi dangereux pour la santé si surconsommé. Ainsi, les paludiers d'aujourd’hui doivent mettre en avant le caractère authentique de leur produit et leur savoir-faire ancestral, symboles de qualité dans l'esprit des contemporains, pour réussir à maintenir leur activité et leur part de marché. L'objectif de cette étude était ainsi de définir des pistes qui garantiraient aux paludiers la protection de leur travail, par exemple dans le cadre d'une démarche d'appellation d'origine contrôlée ou protégée. Pour ce faire, un partenariat avec des sauniers de la Côte Atlantique Française (Ile de Ré, Ile de Noirmoutier, presqu'Ile de Guérande, salines de Saint-Armel) a permis de collecter divers échantillons (eaux des marais, sels). Dans un premier temps, une recherche de microorganismes et une étude sur les traces d'ADNr-16S présent sur les cristaux de sel, ont permis de caractériser une partie du microbiote halophile se développent au niveau des bassins de production du sel de mer, alors que leur teneur en sel peut aller jusqu'à 25 %. Dans un second temps, une recherche de composés volatils a été conduite afin de déterminer si l'environnement pouvait influencer l'empreinte olfactive des eaux des marais et du sel lors de sa formation et / ou de sa récolte. Un protocole d'extraction et d'analyses a été développé et a permis la mise en évidence d'un profil en composés volatils propre à chaque bassin. Parmi les composés volatils détectés, de nombreux norisoprénoïdes, provenant probablement de la dégradation de caroténoïdes produits par les microorganismes halophiles, ont ainsi été identifiés : pour le « bassin » Ile de Ré, 21 composés volatils ont été identifiés dont 8 composés dérivés des caroténoïdes (CDC) ; pour le « bassin » de Noirmoutier, 13 composés dont 7 CDC ; pour le « bassin » de Saint-Armel, 54 composés dont 25 CDC ; pour le « bassin » Guérandais, 19 composés dont 10 CDC.D'une façon générale, les résultats obtenus aussi bien d'un point de vue microbiologique que chimique, ont révélé une forte corrélation entre les marais salants et le sel qu'ils produisent : les microorganismes spécifiques d'un environnement laissent des empreintes sur les eaux et le sel, ce qui permettrait notamment aux paludiers de caractériser leur produit en vue d'une protection basée sur de véritables marqueurs propres à chaque marais (pour des origines distantes de quelques kilomètres, des différences sont déjà notables entre salines tant au niveau « odeur » qu'au niveau microbiote). / Salt has always been exploited in living memory. It has known its heyday at several occasions throughout history, particularly during the period of the French Gabelle, and therefore came to be seen as a precious white metal both sought after and expensive. Yet, nowadays, although it has been considered as an essential mineral, it has also turned up to be unhealthy when taken in excessive amounts. Consequently, salt workers of our day and age have to highlight the genuine nature of their product as well as their ancestral skills, for they both stand out as tokens of quality for our contemporaries. Meeting the expectations of the consumer is the only way for them to keep up with their work and maintain their share on the market. Thus, this study aimed to define ways for salt workers to have their work preserved, for instance throughout a Protected Designation of Origin. Therefore, a partnership with Atlantic French salt workers (from Ré Island, Noirmoutier, Guérande and Saint-Armel) has been established, allowing us to collect samples of salt marsh water and salts.First, an overview of microorganism population and 16S-rDNA in each water or salt sample permitted to define what kinds of microorganism populations were to be found in salt marshes. Secondly, a search for volatile components was led so as to determine whether the environment might affect the olfactory footprint of salt marshes and of salt itself during its formation and its harvest. A process of extraction and analysis has been developed, shedding light on a link between the origin and the olfactory footprint of salt. As an example, the halophilous microorganisms which are extremely rich in carotenoid (hence the red-orange colour of some marshes) are partly responsible for the presence of norisoprenoids in the volatile components which have been identified: 21 compounds were identified in Ré Island (including 8 norisoprenoids), 13 in Noirmoutier (including 7 norisoprenoids), 54 in Saint-Armel (including 25 norisoprenoids),19 in Guérande (including 10 norisoprenoids).For each area, DNA traces and volatile profiles were identified. Therefore, a strong link can be established between salt marshes and the salt they produce. It appears that the differences between salt flats regarding either their smell or their microbiota is always noteworthy, even when marshes are only a few miles apart. Thus, the specific pool of the identified microorganisms which leave prints on the salt would allow saltworkers to define their product so as to ensure a form of protection based on specific markers which are proper to each marsh.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-04T12:10:12Z No. of bitstreams: 1 Dissertação final - Cintia.pdf: 2438772 bytes, checksum: 38cb68fae42cf11ebe768c50e9cb479b (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-03T15:46:44Z (GMT) No. of bitstreams: 1 Dissertação final - Cintia.pdf: 2438772 bytes, checksum: 38cb68fae42cf11ebe768c50e9cb479b (MD5) / Made available in DSpace on 2017-07-03T15:46:44Z (GMT). No. of bitstreams: 1 Dissertação final - Cintia.pdf: 2438772 bytes, checksum: 38cb68fae42cf11ebe768c50e9cb479b (MD5) / As microalgas representam um grupo extremamente diversificado capaz de sobreviver e proliferar em um amplo espectro ambiental. Isto é refletido em sua diversidade e capacidade de modificar o seu metabolismo de forma eficiente em resposta a mudanças ambientais. Além disso, podem ser consideradas verdadeiras fábricas fotossintéticas, sendo fontes potenciais de uma vasta gama de bioprodutos. Dentre os produtos de alto valor agregado e de interesse biotecnológico estão os pigmentos carotenóides que são matérias-primas para indústria farmacêutica, de suplementos alimentares e de cosméticos. Dunaliella salina e Haematococcus pluvialis são exemplos de microalgas com grande potencial de síntese de carotenóides como β-caroteno e astaxantina, respectivamente e, quando submetidas a condições estresse acumulam grandes quantidades no interior das células. Neste trabalho duas espécies de microalgas foram submetidas às condições de estresse por deficiência de nitrogênio e estresse luminoso, com intuito de avaliar a produção dos pigmentos carotenóides em cada condição. Além disso, nosso objetivo também foi conhecer o perfil protéico dessas microalgas em cada condição de estresse a partir de análises por SDS-PAGE. A partir dos resultados, concluiu-se que a privação do nitrogênio no meio de cultura limitou o crescimento das duas microalgas. A microalga D. salina atingiu elevadas concentrações celulares quando cultivada em elevada intensidade luminosa. No entanto, o mesmo não foi observado para microalga H. pluvialis, onde a exposição continua a alta luminosidade atuou de forma negativa ao aumento do número de células. Em relação ao conteúdo de beta-caroteno e astaxantina, a luz contribuiu de forma significativa para o aumento da produção dos pigmentos nas microalgas. No entanto, com privação do nitrogênio no meio, resultou em um menor número de células, consequetemente, a produção de pigmentos pode ter sido influenciada por este fator. Porém, foi observado microscopicamente e visualmente, que a ausência desse elemento possibilitou a produção mais rápida do pigmento nas células, resultando na mudança precoce da cor do cultivo. Avaliando a expressão das proteínas nas microalgas submetidas as condições de estresse, foi observado uma variação de bandas nas diferentes culturas. A expressão de certas proteínas HSPs com diferentes pesos moleculares foi apontada como possível resposta da D. salina e H. puvialis às condições ambientais analisadas. / Microalgae represent an extremely diverse group that can survive and proliferate in a broad spectrum environment. This is reflected in its diversity and ability to modify the metabolism effectively in response to environmental changes. Furthermore, they can be considered true photosynthetic plants, and potential sources of a wide range of byproducts. Among the products of high assembled value and biotechnological interest are the carotenoid pigments that are raw materials for pharmaceuticals, food supplements and cosmetics. Dunaliella salina and Haematococcus pluvialis microalgae are species with potential for the synthesis of carotenoids such as β-carotene and astaxanthin, respectively, and when subjected to stress conditions accumulate large quantities inside the cells. In the present study two species of microalgae were submitted to stress conditions of nitrogen deficiency and light stress, in order to assess the production of carotenoid pigments in each condition. In addition to that, we aimed to know the protein profile of these microalgae in each stress condition from analyzes by SDS-PAGE. From the results it was concluded that the absence of nitrogen in the culture medium limited the growth of both microalgae. The microalgae D. salina reached high cell concentrations when growing in high light intensity. However, this was not observed for microalgae H. pluvialis, since continuous exposure to high brightness limited cell growth. Regarding the content of beta-carotene and astaxanthin, the light has significantly contributed to the increased production of microalgae pigment. However, the deprivation of nitrogen in the medium resulted in a smaller number of cells, consequently, production of pigments may have been influenced by this factor. Although, it was observed visually and microscopically, that the absence of this element enabled faster production of the pigment in cells, resulting in premature color change of the crop. Assessing the protein expression of microalgae subjected to stress conditions, it was revealed a variation in bands in different cultures. The expression of certain HSPs proteins with different molecular weight was highlighted as possible response of D. salina and H. puvialis to the analyzed environmental conditions.

Microalgae, photosynthetic microorganisms, are rich in lipids, polyunsaturated fatty acids, carbohydrates, proteins, vitamins, as well as carotenoids, which are antioxidants that may protect human body from various diseases including obesity, cardiovascular disease, vision-related diseases such as macular degeneration and certain types of cancer. These natural pigments have applications in the pharmaceutical (nutraceutical), food (coloring, functional food, and supplements), and cosmetics industries (e.g. sunscreen), as well as in aquaculture (animal feed). The Dunaliella salina microalga can synthesize 10% of dry weight in &#946;-carotene (orange pigment, pro-vitamin A activity) under high light intensity and nitrogen and phosphorus limitation, among other stress conditions. The first chapter of this thesis presents a review focused on microalgae carotenoids: culture systems, mode of operation, and applications. In this bibliographic survey, the advantages of microalgae cultivation in relation to traditional sources (higher plants) were discussed, as well as a discussion of the main cultivation systems and their importance in cell growth. This review presented a critical analysis of the different operational regimes like batch, fed-batch, semi-continuous and continuous. Relevant information on the most important world producers of microalgae carotenoids were presented. Chapter II presents the development of a modified method of dispersive liquid-liquid microextraction (DLLME) for rapid extraction of &#946;-carotene from Dunaliella salina cultivated in tubular photobioreactor, with subsequent development of a rapid chromatographic screening method using a C4 column for separation of geometric isomer of &#946;-carotene. The use of benzene as extraction solvent and water with 50% acetone as dispersant provided the best condition for the extraction of this carotenoid. In HPLC (High Performance Liquid Chromatography), employing mobile phase composed of methanol and water (95:5, v/v), it was possible to detect/quantify &#946;-carotene at 14 min (retention time). Besides the short analysis time (<20 min), by the miniaturized extraction (< 10 mL organic waste) this method abide by green chemistry analytical principles. It is known that nitrogen, phosphorus, as well as carbon and vitamins are vital elements for the growth of microalgae, also determining the biochemical composition of biomass. In this sense, Chapter III presents the study of the influence of different amounts of sodium nitrate (1N = 75 mg L-1; 1.5N = 112.5 mg L-1, and 3N = 225 mg L-1) and phosphate monobasic dehydrate (1P = 5.65 mg L-1, 1.5P = 8.47 mg L-1, and 3P = 16.95 mg L-1) in seawater-based f/2 medium on the growth of Dunaliella salina and &#946;-carotene biosynthesis, by continuous process with different replenishment proportions (R = 20% and 80%). Best results of cell productivity were obtained by semicontinuous process (mean values of Px up to 6.7 x 104 cells mL-1 d-1 with medium 1N:1P; R =20%) in comparison with batch process cultivation. Maximum cell density (Xm) obtained in this work was not dependent of R, but the best results were obtained when using medium 1.5N:1.5P (mean values up to 5.6 x 105 cells mL-1 with R =80%) instead of 1N:1P. The content of &#946;-carotene in the cells, in general, was higher in cells grown in medium 1N:1P (mean yield values up to 57.5 mg g-1 with R =80%) in comparison with medium 1.5N:1.5P. The cultivation of D. salina with media 3N:3P led to a long lag phase, followed by decrease in cell density and cell lysis. The use of a tubular photobioreactor contributed to successfully cultivate this microalga without contamination by protozoa. The cultivation of Dunaliella salina in tubular photobioreactor with the use of 12:12 photoperiod was appropriate, as well as to induce carotenogenesis, in the second stage, by increasing the light intensity and absence of pH control / As microalgas, micro-organismos fotossintetizantes, são ricas em lipídios, ácidos graxos poli-insaturados, carboidratos, proteínas, vitaminas, além de carotenoides que são antioxidantes com potencial de proteger o organismo humano de várias doenças incluindo a obesidade, doenças cardiovasculares, doenças relacionadas à visão como a degeneração macular e certos tipos de câncer, entre outras. Esses pigmentos naturais têm aplicações em indústrias farmacêuticas (nutracêuticos), alimentícias (colorantes, alimentos funcionais e suplementos) e de cosméticos (exemplo: filtro solar) e na aquacultura (ração animal). A microalga Dunaliella salina é capaz de sintetizar, sob alta intensidade luminosa e limitação de nutrientes como fontes de fósforo e nitrogênio, dentre outras condições de estresse, 10 % do peso seco em &#946;-caroteno (pigmento laranja com atividade pró-vitamina A). Assim, neste trabalho, numa primeira etapa, foi feita uma revisão da literatura abordando a produção de carotenoides por microalgas, bem como sua aplicação. Nesse levantamento bibliográfico abordou-se, dentre outros assuntos, as vantagens do cultivo de microalgas em relação as fontes tradicionais (plantas superiores), assim como uma discussão dos diferentes sistemas de cultivos e sua importância no crescimento celular. Esse review apresentou uma análise crítica dos principais regimes operacionais como batch, fed-batch, semicontínuo e contínuo. Apresentou-se também informações relevantes sobre os mais importantes produtores mundiais de carotenoides de microalgas. Numa segunda etapa, foi desenvolvido um método modificado de microextração líquido-líquido dispersivo modificado (DLLME) para a rápida extração de &#946;-caroteno de Dunaliella salina cultivada em fotobiorreatores tubulares, com subsequente desenvolvimento de método cromatográfico em uma coluna C4 para a separação do isômero geométrico de &#946;-caroteno. A extração ótima de &#946;-caroteno foi obtida com benzeno como solvente extrator e água com 50% de acetona como dispersante. Empregando uma fase móvel composta por metanol e água (95:5, v/v) em HPLC, foi possível a detecção/quantificação de &#946;-caroteno com 14 minutos de tempo de retenção. Além dos tempos curtos de análises (<20 min), pela extração em volume reduzido (< 10 mL resíduos orgânicos) este método obedece aos princípios da química verde. Sabe-se que nitrogênio, fósforo, assim como carbono e vitaminas são elementos vitais para o crescimento das microalgas e também exercem influência na composição bioquímica da biomassa. Assim, na terceira etapa deste trabalho, estudou-se a influência das quantidades de nitrato de sódio (75 mg L-1, denominado 1N; 112,5 mg L-1, denominado 1,5N; 225 mg L-1, denominado 3N) e de fosfato monobásico dihidratado (5,65 mg L-1, denominado 1P; 8,47 mg L-1, denominado 1,5P; 16,95 mg L-1, denominado 3P) em meio f/2, que tem como base a água do mar, no crescimento e na síntese de &#946;-caroteno da Dunaliella salina por processo semicontínuo, com uso de frações de corte (R) de 20% e 80%. Foram obtidas produtividades celulares mais elevadas em processos semicontínuos do que em processo descontínuo, com produtividades médias de até 6,7 x 104 células mL-1 d-1 (meio 1N:1P; R =20%). A máxima concentração celular (Xm) obtida neste trabalho não foi dependente de R. Os melhores resultados de Xm foram obtidos quando se usou meio 1,5N:1,5P em vez de meio, com 1N:1P, com valores médios de até 5,6 x 105 células m L-1 (R =80%). O conteúdo de &#946;-caroteno nas células, de maneira geral, foi maior nas células cultivadas em meio 1N:1P do que no meio 1,5N:1,5P, com valores até 57,5 mg g-1 (R =80%). O cultivo de D. salina com o meio 3N:3P levou a uma longa fase lag, seguida por uma diminuição na concentração celular e sua lise. O cultivo de células em um fotobiorreator tubular contribuiu para um crescimento celular sem contaminação por protozoários. O cultivo de Dunaliella salina em fotobiorreator tubular com o uso de fotoperíodo 12:12 foi apropriado, assim como induzir a carotenogênese, no segundo estágio, por meio do aumento da intensidade luminosa e ausência de controle de pH.

Orientador: Messias Borges Silva / Resumo: O cultivo de microalgas tem sido considerado como uma fonte promissora de lipídeos para a obtenção de ácidos graxos de alto valor agregado, como é o caso dos ômegas 3 (ω3) e ômegas 6 (ω6). O uso do planejamento de experimentos (DOE) permite estabelecer condições apropriadas de cultivo para as microalgas que favorece o acúmulo desses ácidos graxos. Desta forma, o presente trabalho teve como objetivo melhorar o desempenho dos cultivos das microalgas Nannochloropsis gaditana e Dunaliella salina, visando a produção de ω3 e ω6. Primeiramente, foram avaliadas, com o uso de uma matriz ortogonal Taguchi L4, as influências das concentrações de nitrato de sódio (de 25 a 75 mg L-1), de acetato de sódio e bicarbonato de sódio (ambas de 0 a 2 g L-1), sobre a produtividade de biomassa (Pb) e de lipídeos (Po). Como resultado, foi notado que tanto o acetato quanto o nitrato, influenciaram positivamente na Pb e na Po, para ambas as microalgas estudadas. O bicarbonato, por outro lado, não melhorou a Po, sendo excluído das etapas posteriores. Na etapa seguinte do trabalho foi realizado um planejamento fatorial completo 22 com ponto central, focando no estudo da concentração de nitrato (75 a 225 mg L-1) e acetato (2 a 6 gL-1) sobre a produtividade dos cultivos. As maiores Pb obtidas foram: 188,93 mg L-1 dia-1 para a microalga N. gaditana (225 mg L-1 de nitrato e 6 g L-1 de acetato) e 118,93 mg L-1 dia-1 para a D. salina (150 mg L-1 de nitrato e 4 g L-1 de acetato). Nestas condições de cultivo, a... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.