Tesis sobre el tema "Microalgal culture"

A significant proportion of all waste generated in the world is food waste, contributing up to 8% of global CO2 emissions. Conventional ways of food waste disposal including landfilling and incineration are simply inadequate and unsustainable, emphasizing the need for efficient ways recycling / valorizing food waste. Anaerobic digestion is a way to treat and valorize food waste with significantly less emission of greenhouse gases. However, anaerobic digestion itself produces a waste in the form of anaerobic digestate that is difficult to manage. Microalgae cultivation has been used for the treatment and valorization of a wide range of waste effluents and has been identified as a potentially viable option for the treatment and valorization of food waste anaerobic digestate. On the other hand, food waste digestate as a feed stock for the cultivation of microalgae has its challenges. Food waste digestate is a very high strength effluent having ammonia nitrogen concentration of up to 4000 mg L-1, which is extremely toxic to most microalgae species. Food waste digestate is also very turbid, making microalgae cultivation in this effluent extremely difficult without an unsustainably excessive amount of dilution, as light / irradiance is the most limiting factor in any microalgae cultivation system. Previous efforts to treat and valorize food waste digestate have been less than successful due to the reasons stated above. Considering these, we have identified possible solutions to mitigate the challenges involved in treatment and valorization of food waste digestate using microalgae. These are: the identification and application of microalgae species tolerant to high concentration of ammonia nitrogen as found in food waste digestion to tackle the problem of excessive dilution of the digestate before use; and the use of an inclined thin layer pond with a much shorter light path than conventional microalgae cultivation systems allowing for better utilization of incident irradiance in culture systems. Three microalgae (Scenedesmus quadricauda, MUR 268, Chlorella sp, Mur 269, and Oocystis sp, MUR 273) were capable of growth in up to 600 mg L-1 of ammonia nitrogen in food waste digestate. Further experimentation was done at indoor bench scale laboratory conditions using the most tolerant and robust of the 3 identified species, Chlorella sp, Mur 269. To gain insight into the mechanisms required for tolerance of ammonia with the identified species, especially in relation to its response to light profiles imposed by turbidity, their photosynthetic response was studied using pulse amplitude modulation (PAM) fluorometry. Indicators including electron transport rate (ETR), maximum quantum yield in actinic light (fv’/fm’) and alpha (α), alongside oxygenesis as an indicator of photosynthesis, were used to study the state of the photosystem of the organism. Maximum specific growth (μmax) rates and in-situ irradiance profiles were also studied. The effect of turbidity was accounted for by studying the above response variables in clear synthetic medium (Bolds basal medium, BBM) and food waste digestate medium. Maximum specific growth rate (μmax) and biomass productivities were 63% and 47% higher in anaerobic digestate of food waste (ADF) than in BBM, maintaining values of 0.681 ± 0.03 d-1 and 165 ± 8 mg L-1 d-1 respectively, even at high irradiance intensities of 1500 μmol m-2s-1, validating their suitability to outdoor conditions. However, Chlorella sp, Mur 269 photosystem II at optimum irradiance, as reflected in Fv’/Fm’ values, was reduced by 16% in food waste digestate in comparison to BBM. A critical look at the photosynthesis of this algae shows that adaptive tolerance methods of Chlorella sp, MUR 269 to toxicity includes adjustment of the photosynthetic unit to maximize absorption of light and compensation mechanisms for reductions in PSII activity including switching to mixotrophic growth mode. Application of Chlorella sp, Mur 269 for growth in food waste digestate was carried out using an inclined thin layer pond under outdoor conditions. Previous studies using the inclined thin layer pond had shown that though volumetric productivities and biomass densities could be improved by using the inclined thin layer pond for the treatment and valorization of digestates, areal productivities were significantly lower than paddle wheel driven raceway ponds because of the higher surface area to volume ratio in the inclined thin layer pond. To that end, depth optimisation of a 11 m2 surface area inclined thin layer pond was conducted, tailored towards its utilization for the treatment and valorization of food waste digestate using microalgae. Depth optimizations were performed by stepwise increments of the depth of the culture on the surface of the inclined thin layer pond through 0.005, 008, 0.011, 0.0145 and 0.02 m. The kinetics of electron flow around photosystem II of microalgae in-situ culture was used as descriptives for light utilization and limitations of the optimizations via variables including relative electron transfer rate, rETR, and maximum quantum yield, Fv/Fm, and derived parameters including functional relative electron transfer rate (FrETR) and functional relative electron transfer rate ratio (FrETR-ratio). Optimal culture depth determined for the inclined thin layer pond was 0.011 m, with average biomass density of 4.319 ± 0.18 g L-1 and areal productivity of 21.134 ± 1.83 g m-2 d, at an operational volume of 140 L. The most important parameters affecting growth rates and productivity were the mean irradiance inside the culture and the FrETR of photons for phytochemistry. Compared to previous study using anaerobic digestate of piggery waste effluent of similar turbidity, areal productivity was improved 9.5 times. Further, the use of food waste digestate as a source of nutrients for the cultivation of high value species of microalgae such as Dunaliella salina, that are highly sensitive to ammonia nitrogen was explored. The main aim of this study was to assess the possibility of utilizing nutrients from food waste digestate for growing D. salina. Dunaliella salina was cultivated in modified F-medium with partial to complete replacement of the nitrogen (0 – 100 % digestate nitrogen) source under high salinity (150 - 250 ‰) conditions to study both cell growth and carotenogenesis. It was found that the growth and productivity of Dunaliella salina was not inhibited by ammonia nitrogen found in the food waste digestate. Irradiance above 400 μmol photons m-2 s-1 and higher salinity had combined negative effects on growth and carotenogenesis. However, under increased irradiation and temperature, in comparison with cultures grown in synthetic medium with nitrate salts as nitrogen source, there was no significant difference in biomass productivity when D. salina was cultivated using food waste digestate as sole nitrogen source. Finally, a comparative study was performed between the depth optimized inclined thin layer pond and an open raceway pond, both occupying a surface area of 11 m2 under outdoor conditions, to ascertain and demonstrate the advantage of the inclined thin layer pond for the treatment and valorization of food waste digestate using Chlorella sp, Mur 269. The inclined thin layer pond supported a much higher average biomass density of 6.807 ± 0.15 g L-1, 7 times more in comparison to the open raceway pond, without severe photolimitation. Volumetric and areal productivities of the inclined thin layer pond were 0.563 ± 0.1 g L-1 d-1 and 31.916 ± 1.11 g m-2 d-1 respectively, 17 and 3 times higher than observed in the open raceway pond. Areal nutrient removal by the microalgae biomass were 2359.759 ± 64.75 mg m-2 d-1 and 260.815 ± 7.16 mg m-2 d-1 for nitrogen and phosphorous respectively in the inclined thin layer pond, 2.8 times higher than observed in the open raceway pond for both nutrients. The results described above show that employing tolerant species of microalgae like Chlorella sp, Mur 269 offer an advantage for the treatment of high strength effluents, reducing excessive dilution, and unequivocally shows that the inclined thin layer pond is the more performant system for the treatment of highly turbid waste effluents such as food waste digestate.

Due to a number of factors, the biodiesel industry in the United States is surging in growth. Traditionally, oil seed crops such as soybean are used as the feedstock to create biodiesel. However, the crop production can no longer safely keep up with the demand for the growing biodiesel industry. Using algae as a feedstock has been considered for a number of years, but it has always had limitations. These limitations were mainly due to the production methods used to grow and harvest the algae, rather than the reaction methods of creating the biodiesel, which are the same as when using traditional crops. Algae is a promising alternative to other crops for a number of reasons: it can be grown on non arable land, is not a food crop, and produces much more oil than other crops. In this project, we propose a novel attached growth method to produce the algae while recycling dairy farm wastewater using the microalga Chlorella sp. The first part of the study provided a feasibility study as the attachment of the alga onto the supporting substrate as well as determining the pretreatment options necessary for the alga to grow on wastewater. The results showed that wastewater filtered through cheesecloth to remove large particles was feasible for production of Chlorella sp, with pure wastewater producing the highest biomass yield. Most importantly, the attached culture system largely exceeded suspended culture systems as a potentially feasible and practical method to produce microalgae. The algae grew quickly and were able to produce more than 3.2 g/m2-day with lipid contents of about 9% dry weight, while treating dairy farm wastewater and removing upwards of 90% of the total phosphorus and 79% of the nitrogen contained within the wastewater. Once the â proof-of-conceptâ work was completed, we investigated the effects of repeat harvests and intervals on the biomass and lipid production of the microalgae. The alga, once established, was harvested every 6, 10, or 15 days, with the remaining algae on the substrate material functioning as inoculums for repeated growth. Using this method, a single alga colony produced biomass and lipids for well over six months time in a laboratory setting. The second part of this study investigated another aspect of biodiesel production from algae. Rather than focus solely on biomass production, we looked into biodiesel creation methods as well. Biodiesel is created through a chemical reaction known as transesterification, alcoholysis, or commonly, methylation, when methanol is the alcohol used. There are several different transesterification methods. By simplifying the reaction conditions and examining the effects in terms of maximum fatty acid methyl esters (FAME) produced, we were able to determine that a direct transesterification with chloroform solvent was more effective than the traditional extraction-transesterification method first popularized by Bligh & Dyer in 1959 and widely used. This synergistic research helps to create a more complete picture of where algal biodiesel research and development is going in the future.
Master of Science

A promising renewable energy scenario involves utilizing microalgae as biological solar cells to capture the energy in sunlight and then harvesting the biomass for renewable energy production. Through photosynthesis photons are captured by light-sensitive pigment molecules and used to create a cellular chemical energy gradient. Microalgae ultimately use this energy gradient to drive their metabolism by reducing inorganic carbon into renewable, energy-rich organic hydrocarbon stores such as triacylglycerols (TAGs). These valuable molecules act as a cellular energy reserve, readily drawn from when required, often forming large oil-bodies within microalgal cells that can be abundant in certain oleaginous species. This is important for biofuel production because lipids can be extracted from biomass and then converted into a variety of biofuels such as renewable diesel and jet fuel. Thus, from a biofuels perspective, maximizing lipid productivity in selected microalgal feedstock strains is considered essential to the development of an economically viable algal biofuels industry. To achieve this, many current research and development efforts are directed towards genetically engineering well-characterized microalgae to optimize TAG production; however, this approach is a time-consuming, costly prospect and the number of well-characterized strains is relatively few, especially when compared to the number of known extant species. Alternatively, microalgal feedstock optimization could be more readily accomplished by taking advantage of the prodigious natural diversity of microalgae in the environment and identifying native strains of microalgae that, through natural selection, already possess key metabolic traits necessary for commercial feedstock development. Formulated on this premise, a collaborative project between the National Renewable Energy Laboratory (NREL) and the Colorado School of Mines (CSM) recently established and cryopreserved a clonal microalgal culture collection containing 360 unique strains with preliminary data regarding lipid accumulation and the growth potential of select isolates. The goal of this work has been to 1) perform a far more detailed characterization of the algal culture collection by developing high throughput screening procedures and tools for identifying fast-growing, oleaginous strains; and 2) gather further insight into the microalgal diversity found in the southwestern United States. Herein is described in detail the rationale, methods, results and conclusions of these efforts.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A biomassa derivada de microalgas apresenta um grande potencial devido a sua sustentabilidade e alta produtividade, sendo possível extrair lipídios para produção de biodiesel. Entretanto, desafios na cadeia de produção como um todo devem ser resolvidos para que o biodiesel de microalgas seja viável. Uma das etapas críticas é o cultivo, sendo o meio de cultura um elemento de alto custo. O objetivo principal deste trabalho é avaliar o crescimento de microalgas visando produção de lipídios para biodiesel, utilizando como fontes de nutrientes a vinhaça originada do processo produtivo de etanol no setor sucroalcooleiro. Foram isolados e indentificados três gêneros nativos de microalgas: uma cianobactéria Aphanocapsa sp., uma clorofícea Oocystis sp. e outra clorofícea Scenedesmus sp. O cultivo da microalga Scenedesmus sp. em fotobiorreator de placas planas com meio de cultivo MBM (Modified Bristol Medium) se mostrou modesta em termos de produtividade de biomassa (8 mg/l.dia) e em teor de lipídios na biomassa seca (1,5%). O cultivo dessa mesma microalga em tubos de ensaios com meio alternativo utilizando vinhaça (três diluições de 2%, 5% e 10% em volume) no meio de cultura mostrou desempenho comparável em relação ao meio sintético MBM, sendo que a partir do dia 6, os quatros cultivos se estabilizam em torno de uma concentração celular de 6×106 de células/ml, indicando que a vinhaça pode ser uma fonte de nutrientes de baixo custo para o cultivo de microalgas. Deste modo, é possível reduzir custos em uma importante etapa do processo global de produção de biodiesel de microalgas, viabilizando economicamente esta alternativa energética.
The biomass derived from microalgae presents great potential due to its sustainability and high productivity, it being possible to extract lipids to produce biodiesel. However, challenges in the production chain as a whole must be solved to make microalgae biodiesel viable. One of the critical steps is cultivation, with the culture medium being a costly element. The main purpose of this work is to evaluate the microalgae growth focusing in lipid production for biodiesel using as sources of nutrients the vinasse originated from the sugar-alcohol production process. Three natives microalgae genres have been isolated and identified: cyanobacteria Aphanocapsa sp., chlorophycea Oocystis sp. and other chlorophycea Scenedesmus sp. The cultivation of the microalgae Scenedesmus sp. in flat plate photobioreactor with Modified Bristol Medium (MBM) was modest in terms of biomass yield (8 mg/l.day) and lipid content in dry biomass (1.5%). The cultivation of this same microalga in test tubes in an alternative medium using vinasse (three dilutions of 2%, 5% and 10% in volume) in the culture medium showed a comparable performance in relation to the MBM, starting in the 6th day the stabilization of the cell concentration in 6×106 cells/ml for the four cultives, indicating that the vinasse can be a source of low cost nutrients for the cultivation of microalgae. In this way, it is possible to reduce costs at an important stage in the overall process of microalgae biodiesel production, making this energy alternative economically viable.
CAPES: 1420416

High demands in consumer goods and pressures from governments to meet environmental regulations have pushed industries to find innovative, carbon-neutral solutions. Sustainable methods in biotechnology are sought to increase productivity whilst keeping at bay one of the major problems in monoculture production routes: contamination. The use of engineered consortia is seen as a viable option. In nature, microorganisms exist as part of complicated networks known as consortia. Within the consortia, each member plays a role in facilitating communication, tasks distribution, nutrients acquisition and protection. This emerging field uses the conundrums that govern natural microbial assemblages to create artificial co-culture within the laboratory. Purpose fit, co-cultures have been created, to enhance productivity yields of desired products, for bioremediation and to circumvent contamination. The use of microalgae in co-cultures is the focus of this study. Microalgae have application in many fields and are ideal candidates for bioproduction and carbon sequestration. The results of two different systems are presented, which aim to increase the productivity of microalgae biomass and of β-carotene or lipids. The natural consortium of Dunaliella salina, Halomonas and Halobacterium salinarum showed both an increase in microalgae cell concentration by 79% and higher β-carotene productivity compared to the monoculture. This association also showed that Halomonas is able to aid D. salina when subjected to abiotic stress. The artificial co-culture of Scenedesmus obliquus and Rhodosporidium toruloides showed an increase in microalgae biomass by 20%; however, the FAME levels of 26% dw were not a significant increase, compared to monocultures. Both systems demonstrated that if one member of the assemblage is in dire stress, this stress will translate to the entire community. Characterisation of exopolymeric substances and metabolites provided a fuller picture on how these microorganisms co-exist. Additionally, a novel method, duo-plates, was developed and successfully tested to trap metabolites within co-cultures.

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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.

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Financiadora de Estudos e Projetos
The microalgae Haematococcus pluvialis has been studied as one of the main natural sources of astaxanthin carotenoid, potent antioxidant with applications in the nutraceutical and cosmetic industry. H. pluvialis is a microalgae Chlorophyceae whose life cycle includes a phase encystment with high pigment production. In recent years there has been increased activity, processes and applications involving the use of H. pluvialis and its biomass, however H. pluvialis culture do not achieve generally high biomass and species is considered delicate, a slow-growing. Thus, the production and use of the pigment depends on the microorganism and biological, physical and chemical interactions which result in high production of green cells which subsequently form red astaxanthin filled cysts. In this study we sought to optimize the vegetative growth of microalgae, increased the final biomass yield in crops. To this end we investigated the composition of the nutrient medium different pHs and culture methods (sealed vs continuous). Determination of photosynthetic efficiency and energy dissipation were used to infer the cellular health in green flagellates, evaluating the different experimental conditions on microalgae. Intracellular biochemical composition analyzes were performed by determining the concentration of proteins, lipids and carbohydrates in addition to the fatty acid composition. The study was initiated by investigating different nutrient media the growth and biomass production and the results showed that modified Oligo LC medium containing ammonium bicarbonate four times more concentrated nutrients and the remaining 2 times, resulting in improved production of biomass. A procedure then to study the influence of pH on vegetative growth, photosynthetic efficiency and biochemical composition of H. pluvialis. For this purpose we used pH buffers (MES, HEPES, and PIPES), and the results showed a higher germination cysts and higher growth rate in buffered at pH 6.3 cultures (growth rate 0.45 d-1; MES buffer). These findings that the production of H. pluvialis can proceed in the absence of a lag phase in cultures inoculated with cysts. Pigments and lipids related to cell wall dominated at pH 6.3 and palmitic acid (C16:0) and linoleic acid (C18:2n6c) were the most abundant fatty acids. PH in the 6.7 and 7.2 crops showed the highest content of polyunsaturated fatty acids, 6% higher than the control. Regarding the methods of cultivation, continuous were better. The biomass showed higher protein content and the larger culture growth rate and biomass of the sealed. The photosynthetic activity and its parameters suffered significant variations in continuous cultures. H. pluvialis responded better as the photosynthetic parameters in various light intensities when in continuous culture, despite the saturation irradiance was higher in batch cultures.
A microalga Haematococcus pluvialis tem sido estudada por ser uma das principais fontes naturais do carotenoide astaxantina, potente antioxidante com aplicações na indústria de nutracêuticos e cosméticos. H. pluvialis é uma microalga Chlorophyceae cujo ciclo de vida inclui uma fase de encistamento onde o pigmento é acumulado. Apesar de registrar-se um aumento de processos e aplicações envolvendo H. pluvialis, suas culturas dificilmente atingem elevada biomassa e a espécie é considerada sensível a variações ambientais, com crescimento lento. Assim, a produção e uso do pigmento e do microorganismo tornam-se dependentes do desenvolvimento de tecnologia relativas aos fatores biológicos, físicos e químicos, cujas interações resultem em alta produção de células verdes para que posteriormente formem cistos vermelhos repletos de astaxantina. Nesta pesquisa buscou-se otimizar o crescimento vegetativo da microalga, aumentado o rendimento de biomassa final nas culturas. Para isso investigouse a composição de meios nutritivos, diferentes pHs (controle, 6.0, 6.3, 6.7 e 7.2) e modalidades de cultivo (estanque vs contínua). Determinações de eficiência fotossintética e dissipação de energia foram usadas para inferir sobre a saúde celular nas células flageladas verdes, avaliando-se as diferentes condições experimentais. Foram feitas análises da composição bioquímica intracelular determinando-se a concentração de proteínas, lipídios e carboidratos, além da composição de ácidos graxos. O estudo foi iniciado investigando-se diferentes meios nutritivos no crescimento e produção de biomassa e, os resultados mostraram que o meio de cultura LC Oligo modificado contendo bicarbonato de amônio 4 vezes mais concentrado e o restante dos nutrientes 2 vezes, resultou no maior rendimento de biomassa. Procedeu-se então ao estudo da influência do pH no crescimento vegetativo, eficiência fotossintética e composição bioquímica da microalga. Os resultados mostraram maior germinação de cistos e maior taxa de crescimento em culturas tamponadas em pH 6.3 com tampão MES (taxa de crescimento 0,53 d-1). Lipídios relacionados a pigmentos e parede celular dominaram nesse pH e, ácido palmítico (C16:0) e linoleico (C18:2n6c) foram os ácidos graxos de maior abundância. Em relação às modalidades de cultivo, os contínuos foram melhores do que os estanques, que tiveram menor conteúdo proteico e taxa de crescimento. A atividade fotossintética e seus parâmetros sofreu menor variação nas culturas contínuas. H. pluvialis respondeu melhor quanto aos parâmetros fotossintéticos em várias intensidades luminosas quando em cultura contínua, apesar da irradiância de saturação ter sido maior em cultivos estanques.

Esta pesquisa teve como objetivo avaliar a viabilidade do uso da manipueira como meio de cultura para microalgas e determinar as melhores condições para o cultivo. As cepas avaliadas foram isoladas das lagoas de estabilização de manipueira da empresa Podium Alimentos LTDA. de Paranavaí, sendo identificadas morfologicamente como Chlorella sp., Scenedesmus sp. Monoraphidium sp. e Golenkinia sp. A presença de atividade amilolítica e a determinação da toxicidade do cianeto, composto presente no resíduo, foram avaliadas para cada uma das linhagens de microalgas isoladas. A presença de amilase foi identificada em Monoraphidium sp, Golenkinia sp. e Scenedesmus sp. quando inoculadas em meio solido de ágar-amido a 0,2%. A tolerância ao cianeto foi avaliada simulando o ambiente cianogênio com adição de KCN no meio de cultivo autotrófico. As microalgas Monoraphidium sp. e a Scenedesmus sp. demostraram capacidade para se desenvolver em meio contendo até 200ppm de KCN, enquanto as microalgas Chlorella sp. e a Golenkinia sp. suportaram concentrações máximas de 40 ppm. Para avaliação da sobrevivência das microalgas quando cultivadas no resíduo líquido do processamento da mandioca, foi utilizado como meio de cultura manipueira bruta não esterilizada, manipueira bruta estéril e manipueira pré-tratada (digestão anaeróbia) em diferentes concentrações. Os testes foram realizados em frascos Erlenmeyer de 125 mL, sob iluminação de 2500 Lux, fotoperíodo de 12 horas claro/escuro e temperatura de 25°C. As concentrações do inoculo iniciais foram de 0,2 g.L-1. Nos experimentos conduzidos com a manipueira bruta não esterilizada, todas as microalgas mostraram desenvolvimento em concentrações de 10% v/v do resíduo (manipueira/agua). Destacou-se a microalga Monoraphidium sp. que alcançou uma produtividade máxima de 0,014 dia-1, apenas 5% menor que o alcançado quando cultivada em meio autotrófico. Já nos cultivos em manipueira bruta esterilizada as quatro linhagens de microalgas avaliadas apresentaram uma maior resistência ao meio de até 30% de resíduo. Observou-se a maior produtividade com a microalga Monoraphidium sp. e Golenkinia sp. que foi de 0,078 dia-1 e 0,018 dia-1 respectivamente, na concentração de 10% v/v de manipueira. Nos ensaios conduzidos em manipueira após digestão anaeróbia, foram suportadas concentrações de até 100% do resíduo, para o isolado de Monoraphidium sp., Golenkinia sp., e Scenedesmus sp., e de até 40% v/v para a microalga Chlorella sp. A maior velocidade especifica de crescimento foi com a microalga Monoraphidium sp. na concentração de 40% de manipueira (v/v) sendo de 0,12 dia-1. As microalgas Golenkinia sp. e a Scenedesmus sp. também mostraram eficiência quando cultivadas no efluente digerido, sendo que em concentrações de 20% v/v manipueira/água apresentam crescimento semelhante ao obtido no meio sintético. Os resultados obtidos mostraram que é possível o cultivo de microalgas em manipueira bruta, tratada e mesmo manipueira digerida. Porém tratamentos adequados devem ser identificados para se obter uma maior produtividade microalgal. As microalgas Monoraphidium sp. e Scenedesmus sp. demostraram ser capazes de sobreviver e crescer melhor no cultivo em efluente digerido anaerobicamente, assim, a utilização da manipueira digerida como meio de cultivo mostra-se como uma forma eficiente de produzir grandes quantidades de biomassa microalgal.
The objective of this research was to evaluate the viability of the cassava waste water as a culture medium for microalgae and to determine the best conditions for cultivation. The strains evaluated were isolated from the stabilization lagoons of the company Podium Foods LTDA. of Paranavaí, being morphologically identified as Chlorella sp., Scenedesmus sp. Monoraphidium sp. and Golenkinia sp. The presence of amylolytic activity and determination of cyanide toxicity, a compound present in the residue, were evaluated for each of the isolated microalgae strains. The presence of amylase was identified in Monoraphidium sp, Golenkinia sp. and Scenedesmus sp. when inoculated in 0.2% agar-starch solid medium. The cyanide tolerance was evaluated by simulating the cyanogen environment with addition of KCN in the autotrophic culture medium. The microalgae Monoraphidium sp. and Scenedesmus sp. demonstrated the capacity to develop in medium containing up to 200ppm of KCN, while the microalgae Chlorella sp. and Golenkinia sp. have sustained maximum concentrations of 40 ppm. In order to evaluate the survival of microalgae when cultivated in the liquid cassava processing residue, non-sterile cassava waste water handling, sterile manipulative cassava waste water and pre-treated cassava waste water (anaerobic digestion) were used in different concentrations. The tests were carried out in 125 mL Erlenmeyer flasks, under 2500 Lux illumination, 12-hour light/dark photoperiod and 25 °C temperature. Initial inoculum concentrations were 0.2 g.L-1. In the experiments conducted with the non-sterilized raw cassava waste water, all microalgae showed development at concentrations of 10% v/v of the residue (cassava waste water/water). The microalga Monoraphidium sp. which reached a maximum productivity of 0.014 day-1, only 5% lower than that achieved when cultivated in an autotrophic medium. Already in the crops in sterile cassava waste water, the four microalgae strains tested showed a higher resistance to the medium of up to 30% of the residues. The highest productivity was observed with the microalga Monoraphidium sp. and Golenkinia sp. which was 0.078 day-1 and 0.018 day-1 respectively at the 10% v/v concentration of cassava waste water. In the experiments carried out in cassava waste water after anaerobic digestion, concentrations up to 100% of the residue were supported for the isolate of Monoraphidium sp., Golenkinia sp., and Scenedesmus sp., and up to 40% v/v for the microalga Chlorella sp. The highest specific growth rate was with the microalga Monoraphidium sp. in the concentration of 40% of cassava waste water (v/v) being 0.12 day-1. The microalgae Golenkinia sp. and Scenedesmus sp. also showed efficiency when cultivated in the digested effluent, and at concentrations of 20% v/v cassava waste water/water, presented growth similar to that obtained in the synthetic medium. The results showed that it is possible to cultivate microalgae in cassava waste water raw, treated and even digested cassava waste water. However, suitable treatments must be identified for increased microalgae productivity. The microalgae Monoraphidium sp. and Scenedesmus sp. demonstrated to be able to survive and grow better in anaerobically digested effluent cultivation, thus, the use of the digested cassava waste water as a culture medium is shown as an efficient way of producing large amounts of microalgae biomass.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Vinasse is a waste of sugarcane industry and, due to the presence of organic matter and mineral nutrients, it has been used for the irrigation of the sugarcane crops. However, its composition limits such application due to soil salinization. The anaerobic fermentation of vinasse generates biogas that is used as energy source in the sugarcane industry itself. The fermented vinasse results in biodigested vinasse. The development of techniques for residue use can contribute to cleaner production systems. In this research, we evaluated treated conventional and biodigested vinasses for the growth of Chlorella vulgaris. The treatment consisted of filtering the vinasses through smectite clay and activated carbon, or centrifugation. Concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100% of treated vinasses were tested. Cell density, absorbance at 570 nm, pH and electrolyte conductivity were quantified daily in a six day experiment. Based in cell density, specific growth rates were calculated. Growth rates higher than the controls were obtained in 60% conventional filtered vinasse and 10% conventional centrifuged one, as well as in 80% biodigested filtered vinasse and 20% biodigested centrifuged vinasse. In general, the filtered vinasse was superior in relation to the centrifuged one regarding higher residue concentration that supported microalgae growth. After microalgae acclimation we evaluated the photosynthetic performance of C. vulgaris in the best vinasses conditions and the biochemical composition of the biomass present in the vinasse, which besides the microalgae contained other microorganisms, such as bacteria and fungi, since it was not sterilized. Specific growth rate for C. vulgaris in 60% filtered conventional vinasse was 1.6 d-1 and 1.5 d-1 in the 80% filtered biodigested vinasse, representing increases of up to 84 x in cell density compared to the initial. In the LC Oligo control the growth rate was ~ 0.9 d-1 and in the BG11 it was 1,1 d-1. Although the better C. vulgaris growth in the treated vinasses, photosynthetic efficiency and its related parameters were better in the control than in the vinasses. These results together with those of chlorophyll a concentration suggest that the microalgae have possibly used a mixotrophic metabolism while in the vinasses. We observed higher biomass accumulation in the vinasses than in the controls. A reduction of total dissolved organic carbon and dissolved mineral elements up to 50%, as well as bacteria and fungi, were detected in the vinasse after microalgae growth. This study is a contribution to the knowledge of microalgae physiology in a residue and to the development of cleaner ethanol production system.
A vinhaça é um resíduo da indústria sucroalcooleira que, devido à presença de matéria orgânica e nutrientes minerais, tem sido utilizada para fertirrigação das próprias culturas de cana-deaçúcar. Porém, a composição desse resíduo limita sua aplicação no solo, pois pode levá-lo à salinização. A fermentação anaeróbia da vinhaça gera biogás que é usado como fonte de energia para a própria usina. Essa fermentação resulta na vinhaça biodigerida. Técnicas que permitam utilizar as vinhaças, convencionais ou biodigeridas, contribuem para um sistema de produção mais limpo e rentável. Propusemos nesta pesquisa, avaliar o uso de vinhaça tratada. Para tanto usamos vinhaça convencional e biodigerida, filtradas em argila esmectita e carvão ativado, ou centrifugadas. Nelas cultivamos Chlorella vulgaris (BEYERINCK, 1890). As vinhaças tratadas foram testadas em concentrações de 10, 20, 30, 40, 50, 60, 70, 80, 90 e 100%. Avaliamos diariamente a densidade celular, absorbância em 570 nm, pH e condutividade eletrolítica ao longo de seis dias. Com os resultados de densidade celular, calculamos as taxas de crescimento para cada tratamento. Os melhores crescimentos se deram em vinhaça convencional filtrada 60% e centrifugada 10% e, em vinhaça biodigerida filtrada 80% e centrifugada 20%. Em geral, as vinhaças filtradas suportaram o crescimento das microalgas em concentrações superiores em relação às centrifugadas. A produção de biomassa foi maior nos filtrados em comparação com centrifugados. Após aclimatação da microalga em vinhaça avaliaram-se os parâmetros fotossintéticos e bioquímicos da biomassa produzida em vinhaça biodigerida filtrada 80% e em vinhaça convencional centrifugada 60%. Taxas de crescimento específicas de C. vulgaris de 1,6 d-1 foram obtidas na vinhaça filtrada convencional 60% e 1,5 d-1 na biodigerida filtrada 80%, representando aumentos de até 84 x nas densidades celulares em relação às iniciais. No controle LC Oligo esse valor foi de 0,9 d-1 e no BG11 foi de 1,1 d-1. Apesar do melhor crescimento algal nas vinhaças, a eficiência fotossintética e os parâmetros relacionados foram melhores no controle do que nas vinhaças. Esses resultados sugerem que a microalga na vinhaça possa ter utilizado a via mixotrófica como fonte de carbono. Observou-se que houve maior acúmulo de biomassa nos cultivos em vinhaça do que nos controles. Houve redução do conteúdo de carbono orgânico total dissolvido e de elementos minerais na vinhaça em até 50%, bem como da contaminação por fungos e bactérias ao final dos cultivos. Este estudo é uma contribuição para o conhecimento da fisiologia de microalgas em resíduos e para o desenvolvimento de uma produção de etanol mais sustentável.

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A busca por alternativas energéticas viáveis, de baixo custo, sustentáveis e que possam substituir o uso de combustíveis fósseis tem aumentado. Neste contexto, os biocombustíveis derivados de microalgas torna-se uma opção promissora quer seja do ponto de vista econômico como do ponto de vista ambiental. Os cultivos de microalgas produzem elevada quantidade de biomassa em curto tempo e não requerem extensas áreas para sua produção como acontece com a cana de açúcar utilizada para a produção de etanol, ou a soja, no caso do biodiesel. Este trabalho teve o propósito de caracterizar o potencial de produção de ácidos graxos de microalgas isoladas da região semiárida do estado da Paraíba mantidas em cultivo monoespecífico no banco de culturas de microalgas do LARBIM/UFPB e selecionar espécies potencialmente produtoras desses compostos visando cultivá-las em diferentes condições de estresse com o propósito de incrementar o rendimento final dos ácidos graxos de interesse à produção de biodiesel. Os cultivos foram desenvolvidos utilizando os meios Zarrouk e WC em câmara de cultura a 25°C±1ºC dotada de sistema de iluminação, com fotoperíodo de 12 horas. Foram estudadas 12 cepas incluídas em 7 espécies representantes dos grupos das cianobactérias e clorofíceas. A quantificação de ácidos graxos de cada uma das microalgas estudadas, foi determinado por cromatografia a gás. Duas espécies Pediastrum tetras (D121WC) e Scenedesmus acuminatus (D115WC) apresentaram teores de ácidos graxos 802,1% em relação a soja 211,1% em relação a soja, respectivamente. Por essa razão essas espécies foram cultivadas em diferentes condições de estresse nas mesmas condições de temperatura e fotoperíodo, utilizando-se estresses fótico, térmico e nutricional. Ao final de cada experimento foram determinadas para cada condição testada, os teores de protrínas, lipídeos e carbiodratos. As curvas de crescimento foram acompanhas por medida de fluorescência in vivo, em triplicatas, e todos os parâmetros de crescimento foram analisados para cada espécie testada. Observou-se maior taxa de divisão celular por dia na espécie D112Z (0,71 divisão/dia). Os teores de ácidos graxos na cepa D121WC aumentaram mais do que o dobro sob a condição de estresse fótico. Já para a espécie D115WC ocorreu um aumento no rendimento de ácido graxo ao condiciona-la a estresse térmico de 590,8% em relação à soja. Apesar das duas espécies testadas (Pediastrum tetras e Scenedesmus acuminatus) serem potencialmente importantes para a produção de biodiesel, evidenciase que suas respostas fisiológicas às condições de estresse não são as mesmas.
The search for viable energy alternatives, low cost, sustainable and can replace the use of fossil fuels has increased. In this context, biofuels derived from microalgae become a promising option either from the economic point of view as from the environmental point of view. The cultivation of microalgae produce higher amount of biomass in a short time and require large areas for their production as with sugarcane used for ethanol production, or soybean in the case biodiesel. This study aimed to characterize the potential for production of fatty acids from microalgae isolated from the semiarid region of the state of Paraiba kept growing in monospecific cultures of microalgae bank of LARBIM / UFPB and select potentially producing species of these compounds in order to grow them different stress conditions in order to increase the final yield of the fatty acids of interest for biodiesel production. The cultures were developed using the means Zarrouk and toilet in a growth chamber at 25 ° C ± 1 ° C equipped with lighting system, with a photoperiod of 12 hours. 12 strains included representatives of groups of cyanobacteria and green algae were studied in seven species. The quantification of each fatty microalgae studied acids was determined by gas chromatography. Two species Pediastrum tetras (D121WC) and Scenedesmus acuminatus (D115WC) showed levels of fatty acids 802.1% compared to 211.1% in soybeans compared to soybean, respectively. Therefore these species were grown under different conditions of stress under the same conditions of temperature and photoperiod, using photic, thermal and nutritional stresses. At the end of each experiment were determined for each condition tested, the levels of proteins, lipids and carbohydrates. The growth curves were avidly by measuring in vivo fluorescence in triplicate, and all growth parameters were analyzed for each species tested. Observed a higher rate of cell division per day in D112Z (0.71 divisions / day) species. The levels of fatty acids in strain D121WC increased more than twice on condition of photic stress. As for the species D115WC was an increase in the yield of fatty acid to condition it to heat stress of 590.8% compared to soybean. Although the two potentially important for the production of biodiesel tested species (Pediastrum tetras and Scenedesmus acuminatus) are, it is evident that their physiological responses to stress conditions are not the same.

Orientador: Ricardo Alan Verdú Ramos
Resumo: A biomassa derivada de microalgas apresenta um grande potencial devido a sua sustentabilidade e alta produtividade, sendo possível extrair lipídios para produção de biodiesel. Entretanto, desafios na cadeia de produção como um todo devem ser resolvidos para que o biodiesel de microalgas seja viável. Uma das etapas críticas é o cultivo, sendo o meio de cultura um elemento de alto custo. O objetivo principal deste trabalho é avaliar o crescimento de microalgas visando produção de lipídios para biodiesel, utilizando como fontes de nutrientes a vinhaça originada do processo produtivo de etanol no setor sucroalcooleiro. Foram isolados e indentificados três gêneros nativos de microalgas: uma cianobactéria Aphanocapsa sp., uma clorofícea Oocystis sp. e outra clorofícea Scenedesmus sp. O cultivo da microalga Scenedesmus sp. em fotobiorreator de placas planas com meio de cultivo MBM (Modified Bristol Medium) se mostrou modesta em termos de produtividade de biomassa (8 mg/l.dia) e em teor de lipídios na biomassa seca (1,5%). O cultivo dessa mesma microalga em tubos de ensaios com meio alternativo utilizando vinhaça (três diluições de 2%, 5% e 10% em volume) no meio de cultura mostrou desempenho comparável em relação ao meio sintético MBM, sendo que a partir do dia 6, os quatros cultivos se estabilizam em torno de uma concentração celular de 6×106 de células/ml, indicando que a vinhaça pode ser uma fonte de nutrientes de baixo custo para o cultivo de microalgas. Deste modo, é possível r... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Mestrado em Biotecnologia Industrial e Ambiental
As microalgas são uma fonte emergente de alimento, corantes alimentares, combustível e produtos químicos. O estudo dos processos upstream e downstream são de extrema relevância pois de modo a prever a sua exploração industrial. O presente trabalho, está dividido em dois capítulos, um envolvendo o processamento upstream e outro explorando o processamento downstream. A microalga M, está há muito documentada pelo seu potencial como fonte para produção de biopolímeros contudo o cultivo outdoor desta microalga está ainda por otimizar. Desta forma, o estudo do efeito de parâmetros operacionais de cultivo, nomeadamente D, E e F, foram usados para desenvolver três modelos matemáticos em relação a A, B e C para culturas continuas de microalga X em escala de laboratório. Os resultados mostraram ser possível atingir culturas com elevadas A, elevado conteúdo de C ou B com tamanho controlável, através da manipulação das condições de cultivo. Com aplicação do presente estudo é esperado que se aumente a transição de conhecimento de cultivo reduzida para larga escala, de forma a potenciar a aplicação de larga escala. O capitulo de processamento downstream no presente trabalho visa o desenvolvimento de uma metodologia alternativa de extração de ficocianina em culturas de Anabaena cylindrica. Os líquidos iónicos são uma classe de solventes que têm atraído muita atenção por parte dos investigadores pelas suas características únicas. O uso de soluções aquosas de líquidos iónicos mostrou-se altamente seletivo para a extração de ficocianina e clorofila. A performance da solução aquosa de [C4mimCl] foi otimizada quando os parâmetros operacionais de extração foram: rácio solido-liquido 0,1 (peso de biomassa fresca/volume de solvente), tempo de extração (43 min.) e concentração do liquido iónico (475 mM). Sob estas condições os rendimentos de ficocianina e grau de pureza alcançados foram 55 ± 2 mgficocianina.gbiomassa fresca-1 e 0,54, respetivamente. O uso de soluções aquosas de liquido iónicos resultou num extrato bruto rico em ficocianina com reduzida contaminação de outros pigmentos e outras proteínas. Assim esta metodologia alternativa demonstrou-se mais efetiva do que outras tentativas encontradas na literatura ao passo que tem a capacidade de simplificar os processos de purificação e concentração, comumente reconhecidos por serem dispendiosos e dificilmente escaláveis.
Microalgae has been regarded as an emerging source for feed, food colourants, fuel and chemicals. The study of the upstream and downstream processing is of foremost importance in order to foresee its industrial exploitation. Therefore, the present work is divided in two chapters, one comprising the upstream and another the downstream processing. The potential of Microalgae M as a source of biopolymers is already known though its outdoor cultivation is still to optimize. Thus, the effect of relevant environmental parameters, namely D, E and F were studied and used to develop three mathematical models regarding the A, B and C for continuous cultures of Microalgae X at lab scale. The results show that it’s possible to achieve increased A, high content of C or B with wieldy size through the manipulation of these culture conditions. The application of the present study is expected to fill the gap between lab and large-scale cultures to enhance and optimize its large-scale performance. The downstream processing chapter in the present work regards the development of an alternative extraction methodology to recover a natural pigment, the phycocyanin from Anabaena cylindrica cultures. Ionic Liquids are a class of solvents that have been attracting much attention from researchers due to its unique properties. The use of aqueous solutions of ionic liquids has shown highly selective performances for phycocyanin and chlorophyll extraction. The performance of aqueous solutions of an imidazolium-based ionic liquid, namely the [C4mim]Cl was optimized after the operation extraction parameters were set: solid-liquid ratio of 0.1 (weight of fresh biomass/volume of solvent), extraction time (43 min) and ionic liquid concentration (475 mM). Under these conditions phycocyanin yields and purity levels (A615/A280) were 55 ± 2 mgphycocyanin.gfresh biomass-1 and 0.56, respectively. The use of aqueous solutions of ionic liquids resulted in crude extracts rich in phycocyanin with reduced levels of pigments and proteins contamination, whereas it was shown more effective than other attempts found in literature as well as it can simplify the purification and concentration steps, commonly known for being expensive and hardly-scalable.

Although, microalgal wastewater treatment systems represent an efficient and economical alternative to conventional processes, the use of marine microalgae to remove nutrients from wastewaters has not been extensively researched and few studies have been made in temperate and high latitude areas, where climate may limit treatment. In this study, 102 marine microalgal species, including 66 local endemic isolates from St Andrews Bay, Fife, Scotland, were screened under batch and continuous culture. Species were selected for their ability to remove high concentrations of ammonium and ortho-phosphate from primary treated sewage (diluted 1:1 with sterile seawater) while remaining dominant in culture. Abiotic removal of ammonium and ortho-phosphate at high pH was found to be low from saline media, indicating that much of the nutrient removal from the seawater:wastewater mixture was by algal uptake. Many of the best-treating species grew over a wide range of temperature (10-25 °C), and their growth was not inhibited by the low salinity of the 1:1 diluted wastewater. Seven best-treating species continuously removed >80 % ammonium and >70% ortho-phosphate when cultured in 20 litre mini-ponds (modelled on high-rate ponds) under ambient summer conditions over two weeks. These were all endemic isolates including six bacillariophyceaen isolates (of which three were strains of Phaeodactylum tricornutum), and a species of the cyanophyceaen Oscillatoria. Two isolates (Oscillatoria and an unidentified bacillariophyceaen SA91B33) with adherent properties, continuously removed 100 % of both ammonium and ortho-phosphate when tested in a corrugated raceway designed to provide a large surface area for attachment. Preliminary experiments further showed the best-treating species to be capable of removing nutrients from eel aquaculture effluent. The abilities of marine microalgal species to remove high concentrations of nutrients, remain in unialgal culture and grow over a range of environmental conditions are indicative of their potential for use in wastewater treatment systems in temperate areas.

Current abattoir wastewater treatment options involving aerobic and anaerobic digestion systems are relatively inefficient in reducing the inorganic nutrient load (i.e. ammonium and phosphate) of the effluent. With anaerobic lagoons favoured in Australia resulting in large land footprint, loss of environmental and economic value in the form of water and nutrient recovery. The cultivation of microalgae on undiluted anaerobic digestion abattoir effluent (ADAE) offers many potential benefits such as the bioremediation of waste nutrients typically found in the effluent and simultaneous production of valuable algal biomass which represents the conversion of waste-to-profit. Microalgal culture on undiluted ADAE require minimal to no freshwater input and does not compete for arable land, it has the potential to play an important remediation role particularly during the final (tertiary) treatment phase. The generated biomass can also be a source of revenue for the targeted abattoir. To the best of my knowledge, to date, there has been no attempt on testing the growth of algae to treat undiluted ADAE. In this study, the growth, biomass productivity, photo physiology and nutrient removal rates of mono and mixed culture of Chlorella sp. and Scenedesmus sp. were evaluated in unfiltered and undiluted ADAE. Chlorella sp. showed the highest ammonium removal rate of up to 11.93±1.14 mgL-1 d-1 as well as the highest biomass productivity of 31.52±0.81 mgL-1 d-1 when compared to the other treatments. Phosphate removal rates were highest in the Scenedesmus cultures while the mixed cultures varied amongst other parameters measured (COD, Nitrite and Nitrate). Chlorella sp. with a specific growth rate of 0.260±0.047 d-1 and mixed Chlorella sp. within consortium (0.294±0.024 d-1) were the dominant species when compared to Scenedesmus sp. (0.062±0.011 d-1). Overall, Chlorella sp. was identified as the most efficient microalgal species capable of growing on undiluted ADAE with the potential to generate a waste-to-profit system.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O estudo do cultivo de microalgas na aquicultura é importante para incrementar o conhecimento da biologia das espécies, favorecendo posterior produção em ambientes controlados. O objetivo deste trabalho foi avaliar o crescimento da microalga Ankistrodesmus gracilis em meio de cultura composto por biomassa de Azolla caroliniana e Lemna minor e utilizar a microalga na alimentação do Xiphophorus maculatus visando melhorar o desempenho zootécnico. O experimento com a microalga durou 28 dias em condições controladas e os parâmetros físicos e químicos foram mensurados semanalmente com a avaliação diária do crescimento. A planta L. minor apresentou melhores resultados para macro e micronutrientes, e o crescimento também foi maior no meio de cultura para a microalga A. gracilis (552 x 105 cel mL-1) do que no meio de A. caroliniana (292 x 105 cel mL-1). No meio de cultura de L. minor a microalga A. gracilis apresentou maiores teores de clorofila-a, taxa de crescimento, densidade celular média e máxima e fósforo total. O teor de lipídio foi maior no meio de L. minor e o de proteína foi igual em ambos os meios. O uso de microalga A. gracilis crescida no meio de macrófita, foi utilizada para a alimentação do peixe ornamental Platy (X. maculatus). Para preparação da ração a microalga crescida nos dois meios de cultura diferentes foi liofilizada e adicionada na ração na proporção de 10%. Os tratamentos foram alimento inerte, somente ração (controle); alimento inerte com microalga A. gracilis cultivada em meio de cultura A. caroliniana e alimento inerte com microalga A. gracilis cultivada em meio de cultura L. minor. Em relação ao desempenho zootécnico observou-se diferenças significativas (p<0,05) para o tratamento contendo microalga cultivada em meio de L. minor em relação ao peso final, ganho de peso, taxa de crescimento, consumo, peso inicial e sobrevivência, e o controle obteve o pior desempenho zootécnico. Dentre as duas macrófitas estudadas L. minor demonstrou ser mais eficiente promovendo elevada densidade celular, evidenciando que estas plantas podem ser uma nova ferramenta para o cultivo em laboratório desta microalga, em função do baixo custo, eficiência de produção e disponibilidade do recurso. Para o peixe ornamental X. maculatus a ração contendo a microalga cultivada em meio de cultura L. minor apresentou melhores resultados. Já no de A. caroliniana apesar de inferior a L. minor demonstrou ser melhor que o controle (somente ração), comprovando que as macrófitas além de servirem como meio de cultura para microalga, também podem ser um incremento em rações de peixes ornamentais.
Studies on the cultivation of microalgae are greatly relevant for in-depth analysis of species´ biology and for highlighting production in controlled environments. Current analysis assesses growth of the Ankistrodesmus gracilis microalgae in a culture medium composed of Azolla caroliniana and Lemna minor biomass and to use a microalga in the feeding of Xiphophorus maculatus aiming to improve the zootechnical performance. Assay with microalga lasted 28 days in controlled conditions, and physical and chemical parameters were measured weekly, coupled to daily growth evaluation. Macrophyte L. minor had better results macro and micronutrients. and growth was also higher in the culture medium for microalgae A. gracilis (552 x 105 cell mL-1 ) than in A. caroliniana medium (292 x 105 cell mL-1 ). Microalga A. gracilis had higher rates of chlorophyll- , growth and medium, maximum cell density rates and total phosphorus in this culture medium. Lipid rates were greater in L. minor medium. Protein rate was equal in the two media. The use of microalga A. gracilis grown in macrophyte medium was employed for the feeding of the ornamental fish Platy (X. maculatus). To prepare the feed the microalgae grown in the two different culture media were lyophilized and added to the ration in the ratio at 10%. Treatments consisted of inert feed, ration only (control); inert feed plus microalga A. gracilis cultivated in A. caroliniana medium; inert feed plus microalga A. gracilis cultivated in L. minor medium culture. In the case of animal performance, significant differences (p<0.05) were registered for treatment with microalga cultivated in L. minor medium with regard to final weight, weight gain, growth rate intake, initial weight and survival, and the control obtained the worst zootechnical performance. L. minor proved to be more efficient since it enhanced high cell density. This fact showed that these plants may be a new tool for laboratory culture due to low costs, production efficiency and availability. In the case of the ornamental fish X. maculatus, feed with the microalga cultivated in L. minor medium had the best results. In the case of A. caroliniana, albeit lower than L. minor, it proved to be better than control (feed only). Besides being employed as culture medium for microalgae, macrophytes may also constitute a partial increase in the ration of ornamental fish.
134543/2016-5

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A biotecnologia utilizada no cultivo de microalgas à base de meio comercial é de alto custo. Sendo assim, alguns estudos indicam que o uso de meios de cultura alternativos com a finalidade de reduzir o preço de produção em curto espaço de tempo e mantendo alto valor nutricional, são procedimentos a serem adotados. O objetivo deste trabalho foi avaliar a qualidade de diferentes meios de cultivo a base de macrófitas. O meio com macrófita foi associado ao fertilizante inorgânico NPK (20:5:20). O experimento foi conduzido no período de 28 dias em triplicata. O meio de cultura com E. crassipes alcançou maior densidade celular (434 x 105 cel. mL-1), comparado com os outros meios de cultura com macrófitas que variaram de 319,7 x 105 cel. mL-1 (E. azurea) a 223,5 x 105 cel. mL-1 (T. domingensis). Durante o período experimental o oxigênio dissolvido, pH e condutividade elétrica não apresentaram diferenças significativas (P>0,05). A taxa de crescimento e densidade celular da microalga cultivada em meio de cultura com plantas aquáticas foram maiores (P>0,05) do que no meio NPK porém, similares (P<0,05) ao meio comercial CHU12. Em relação aos nutrientes, o teor de N (67 g.L-1) foi o mais elevado no meio de cultura com T. domingensis e os demais nutrientes (P, K, Ca, S, B, Cu, Fe e Zn) apresentaram concentrações abaixo de 35 g.L-1, nos diferentes meios utilizados. O uso de macrófitas como fonte alternativa para ser utilizada como meio de cultura no desenvolvimento de A. gracilis demonstrou ser viável tanto do ponto de vista econômico, nutricional e com elevada biomassa algal, sendo que E. crassipes, E. azurea e T. domingensis apresentaram os melhores resultados.
Since biotechnology in commercial medium-based culture of microalgae has very high costs, several studies recommend alternative medium cultures to reduce product costs in a short time period, coupled to high nutritional values. Objective of this work was to evaluate the quality of different macrophytes based culture media. Medium with macrophyte was associated with inorganic fertilizer NPK (20:5:20). Assay was conducted for 28 days, in triplicate. Culture medium with E. crassipes had the greatest cell density (434 x 105 cel. mL-1) when compared to other culture media with macrophytes and ranged between 319.7 x 105 cel. mL-1 (E. azurea) and 223.5 x 105 cel. mL-1 (T. domingensis). Dissolved oxygen, pH and electric conductivity did not show any significant differences during the experimental period (p>0.05). Growth rate and cell density of microalga cultivated in a medium with aquatic plants were greater (p>0.05) than in NPK medium, albeit similar (p<0.05) to the commercial medium CHU12. Further, N rate (67 g.L-1) was the highest in culture medium with T. domingensis, whilst the other nutrients (P, K, Ca, S, B, Cu, Fe and Zn) had lower than 35 g.L-1 concentrations in the different media employed. The use of macrophytes as an alternative source as culture medium in the development of A. gracilis proved to be viable from the economic and nutritional point of view, coupled to high algal biomass. E. crassipes, E. azurea and T. domingensis provided the best results.
CNPq: 130584/2015-0
FAPESP: 2014/24697-3

Background and Aim: Microalgae are a potential economical source of omega-3 fatty acids, which are known for their health benefits. Omega-3 fatty acids derived from microalgae are however still expensive due to high costs involved in production. These costs can partly be attributed to cultivation of microalgae in unialgal cultures, which are prone to contamination, difficult to scale up and require stringent control of growth conditions. This study therefore is aimed to investigate the feasibility of using a mixed microalgae culture for the production of omega-3 fatty acids. Methods and materials: The microalgae were grown under uncontrolled conditions in a hanging-bag photo-bioreactor, with only the phosphorus-nitrogen ratio (P:N-ratio) varied. Variations in the different population proportions in the culture were studied and related to the biomass, total fatty acid and omega-3 fatty acid productivity of the culture. Finally, a comparison was made between the results obtained in this study and similar studies found in literature. Results and discussion: The mixed culture used was composed of several green microalgae, whose proportions varied with P:N-ratio, season, and age of the culture. Different interactions between the populations were observable, and these were thought to influence the culture’s biomass, total fatty acids (TFAs) and omega-3 fatty acid productivity. The TFA content of the culture was ~10-20% by the end of the exponential phase, depending on the nutrients supplied. The fatty acids were composed of mostly unsaturated fatty acids, the majority of which were omega-3 fatty acids; C18:3ω3 (up to 50% of TFA), C16:4ω3 (up to 15%) and C18:4ω3 (up to 5%). Other fatty acids observed included C16:0 (up to 25%), C18:1ω9/C18:1ω8 (up to 20%), C18:2ω6 (up to 5%), traces of C18:0, C18:3ω6, C17:0, C16:3ω3 and C16:1ω7 were also detected. The presence of these fatty acids was found to vary depending on season, P:N-ratio, and age of the culture. When compared to studies in the literature, the mixed culture was found to perform better than, or as well as reported studies, where unialgal cultures were cultivated under controlled laboratory conditions. Conclusion: In view of the reported advantages of cultivating mixed cultures, the production of omega-3 fatty acids via such cultures appears attractive. More research however is needed to optimise the culture; study interactions between the organisms and their effect on biomass, total fatty acid and omega-3 fatty acid productivity. Finally, the economic implications of using mixed cultures need to be evaluated as well.

Three local isolates of the green alga Tetraselmis sp. identified as the most promising microalgae species for outdoor mass cultivation with high potential for biodiesel production due to high amounts of total lipids and high lipid productivity were employed in this study. The aim of the study was to compare three halophilic Tetraselmis strains (Tetraselmis MUR-167, MUR-230 and MUR-233) grown in open raceway ponds over long periods with respect to their specific growth rate and lipid productivity without additional CO2 and with CO2 addition regulated at pH 7.5 by using a pH-stat system. Attention also was given to the overall culture condition including contaminating organisms, biofilm development due to cell adhesion and cell clump formation. All tested Tetraselmis strains in this study were successfully grown outdoors in open raceway ponds in hypersaline fertilised medium at 7 % w/v NaCl over a period of more than two years. A marked effect of CO2 addition on growth and productivities was observed at high solar irradiance and temperatures between 15 – 33 oC. However, differences were identified between the three Tetraselmis strains in biofilm development, specific growth rate and lipid productivity in association with solar irradiance and temperature. Study of Tetraselmis MUR-167 was terminated after two years of outdoor cultivation due to stickiness and biofilm development on the pond walls, with a consequent decline in specific growth and productivities. Tetraselmis MUR-230 and MUR-233 showed quite similar annual specific growth and average productivities of 12 g (ash free dry weight) m-2 d-1 and 5.3±0.4 g m-2 d-1 in semi-continuous culture over a period of 365 days for biomass and lipid respectively; however MUR-233 had a higher specific growth rate and higher lipid productivity at higher temperatures in comparison with MUR-230. Further studies were carried out on Tetraselmis MUR-233 in 2 m2, 20 m2 and 200 m2 raceway ponds at a pilot plant in Karratha, West Australia. Comparison between Perth and Karratha showed that Tetraselmis MUR-233 had up to three times higher biomass productivities in winter and 40 % higher during summer in Karratha. Appreciable differences in specific growth during semicontinuous cultivation were observed in 2 m2 raceway ponds compared to the 20 m2 and 200 m2 ponds accompanied with a decline in biomass productivity of up to 20 % with increasing pond size. Highest biomass productivities of 30 g (ash free dry weight) m-2 d-1 and a lipid content of up to 43 % based on ash free dry weight was achieved at an optimum cell density of 60 x 104 cells .mL-1 combined with 0.4 daily dilution and CO2 addition even under hyper saline conditions. These results were obtained at 25 MJ .m-2 solar irradiance and temperatures between 15 – 31 oC. Tetraselmis MUR-230 and MUR-233 with CO2 addition and regulated pH out-competed the major contaminating organisms, the diatoms. Tetraselmis MUR-230 grew successfully with different N-sources such as ammonium chloride, urea and sodium nitrate, the highest cellular lipids obtained with urea and sodium nitrate. Cellular carbohydrates in Tetraselmis MUR-230 increased five times under N-depletion and CO2 addition. In summary, this study demonstrates the feasibility of outdoor large scale cultivation of the halophilic Tetraselmis strains MUR-230 and MUR-233 with a high potential as feedstock for biofuel and carbon dioxide mitigation. The sustainability is underlined by the fact that the Tetraselmis strains were able to grow in hot and dry areas using only seawater as water source.

Due to the large volume of the processed medium in pilot or industrial cultivation systems, it is difficult to illuminate the entire layer of the culture medium, which results in the formation of dark zones. Due to insufficient mixing, also an unbalanced utilization of nutrients contained in the culture medium or formation of temperature gradients can occur. Microalgal biofilm formation attached to the transparent walls of closed photobioreactors (PBRs) is also a significan! limitation associated with scaling-up, since it can significantly reduce the intensity of incident light. According to those factors, the hydrodynamic conditions of the culture medium are an importan! parameter in the scaling-up of cultivation systems, since it affects the mixing and the homogeneity of the culture medium. Efficient mixing can: 1) allow all microalgal cells to reach the irradiated area (light zone) of the culture medium; 2) prevent the formation of temperature gradients or sedimentation of microalgal cells; 3) intensify mass transfer resulting in more efficient utilization of nutrients. Moreover, the intensification of flow in the a rea clase to the transparent walls of the cultivation system can also result in an in crease of wall shear stress and a reduction of biofilm formation. The aim of this thesis was to study the influence of hydrodynamic conditions on parameters affecting the production of microalgae in two cultivation systems: a hybrid horizontal tubular photobioreactor (HHT PBR) and a closed flat panel photobioreactor (FP PBR). To this end, a multi-physical model was created to study the effect of hydrodynamic conditions on microalgae cultivation. Based on the experimental measurements, a numerical model simulating the hydrodynamic conditions in transparent HHT PBR tubes was validated. Through the model, the influence of different operating conditions on the mixing of the culture medium were investigated. The developed multi-physical model allowed to investigate the influence of operating conditions on the distribution of light in the culture medium and the production of microalgae, The model showed that, in systems working with a large !ayer of culture medium or a high concentration of microalgae, the intensification of mixing increases the production of microalgae. To do this, the hydrodynamic conditions in the cultivation system should be brought as clase as possible to the state where the entire volume of the culture medium is ideally mixed. This state can be achieved by increasing the flow rate in the tubes or by using static mixers installed in the tubes of the PBR. Hydrodynamics in FP PBR were more complex than in HHT PBR. By comparing the created ydrodynamic model with experimental measurements, the influence of hydrodynamics on the prevention of biofilm formation was specified as well. The created hydrodynamic model allowed to optimize the operating and design parameters of the FP PBR. In arder to intensify the mixing and homogenize the flow of the culture medium in the FP PBR, a static mixer was designed. Compared to the empty FP PBR chamber, the homogenization time was reduced and the homogenous flow in the chamber was ensured by the installed static mixer The multi-physical model developed in this thesis has preved to be an efficient tool to understand the influence of hydrodynamic conditions on microalgae production. Based on experimental measurements and numerical models, the operating conditions of HHT PBR and FP PBR were optimized. To further intensify the mixing and homogenize the flow of the culture medium, a static mixer was designed, which demonstrated a positive effect on the hydrodynamic conditions of the culture system. Overall, the created numerical model is a useful tool to improve existing cultivation systems, to acquire knowledge during the scale-up of cultivation systems or for designing novel PBRs.
Debido al gran volumen de los sistemas piloto o industriales, es difícil iluminar todo el volumen del medio de cultivo, lo que da lugar a la formación de zonas en sombra. También puede producirse una utilización desequilibrada de los nutrientes contenidos en el medio de cultivo o la formación de gradientes de temperatura si la mezcla es deficiente. La formación de biopelículas de microalgas adheridas a las paredes transparentes de fotobiorreactores cerrados (PBR) es también una limitación significativa asociada con el escalado, ya que puede reducir significativamente la intensidad de la radiación de luz incidente. De acuerdo con estas limitaciones, un factor determinante en el escalado de los sistemas de cultivo son las condiciones hidrodinámicas en el ismo, ya que afectan al mezclado y la homogeneidad del medio de cultivo. Una mezcla eficiente puede: 1) permitir que todas las células alcancen el volumen iluminado del medio de cultivo; 2) prevenir la formación de gradientes de temperatura o la sedimentación de células; 3) intensificar la transferencia de masa dando como resultado una utilización más eficiente de los nutrientes. Además, la intensificación del flujo en la zona cercana a las paredes transparentes del sistema de cultivo da lugar a un aumento del esfuerzo cortante en la pared y una reducción de la formación de biopelículas. El objetivo de esta tesis ha sido estudiar la influencia de las condiciones hidrodinámicas sobre los parámetros que afectan a la producción de microalgas en dos sistemas de cultivo: un fotobiorreactor tubular horizontal híbrido (HHT PBR) y un fotobiorreactor cerrado de placa plana (FP PBR). Con este fin, se construyó un modelo multifísico para estudiar el efecto de las condiciones hidrodinámicas en el cultivo de microalgas. Se construyó un modelo numérico que simula las condiciones hidrodinámicas en tubos transparentes HHT PBR, el cual fue validado a partir de mediciones experimentales. Mediante el modelo, se investigó la influencia de diferentes condiciones operativas en la mezcla del medio de cultivo. El modelo multifísico desarrollado permitió evaluar la influencia de las condiciones operativas en la distribución de la luz en el medio de cultivo y la producción de microalgas. El modelo mostró que, en sistemas que trabajan con una gran profundidad de medio de cultivo o una alta concentración de microalgas, la intensificación de la mezcla aumenta la producción de microalgas. Para ello, las condiciones hidrodinámicas en el sistema de cultivo deben aproximarse lo más posible al estado de mezcla ideal en todo el volumen del medio de cultivo. Este estado se puede lograr aumentando el caudal circulante en los tubos o utilizando mezcladores estáticos instalados en los tubos del PBR. La hidrodinámica en el FP PBR es más compleja que en el HHT PBR. Al comparar las simulaciones del modelo hidrodinámico con mediciones experimentales, también evaluó la influencia de la hidrodinámica en la prevención de la formación de biopelículas. El modelo hidrodinámico desarrollado permitió optimizar los parámetros operativos y de diseño del FP PBR. Para intensificar el mezclado y homogeneizar el flujo del medio de cultivo en el FP PBR, se diseñó un mezclador estático. En comparación con la cámara del FP PBR vacía, mediante la instalación del mezclador estático se redujo el tiempo de homogeneización y se aseguró el flujo homogéneo en la cámara. El modelo multifísico desarrollado en esta tesis ha demostrado ser una herramienta eficaz para comprender la influencia de las condiciones hidrodinámicas en la producción de microalgas en fotobiorreactores. Basándose en mediciones experimentales y modelos numéricos, se han optimizado las condiciones de funcionamiento de HHT PBR y FP PBR. Para intensificar aún más la mezcla y homogeneizar el flujo del medio de cultivo, se diseñó un mezclador estático, que demostró tener un efecto positivo sobre las condiciones hidrodinámicas del sistema de cultivo. En definitiva, el modelo numérico creado es una herramienta útil para optimizar la operación de los sistemas de cultivo existentes, para adquirir conocimientos durante el escalado de los sistemas de cultivo y para diseñar nuevos PBR.
(Txec) Mezi nejdůležitější provozní parametry zajišťující správnou kultivaci mikrořas patří světelné záření, vhodná teplota kultivačního média, koncentrace živin a CO2 v kultivačním médiu. Při zvětšování měřítka kultivačních systémů jsou však právě tyto parametry limitujícím faktorem. Vzhledem k velkému objemu zpracovávaného média v poloprovozních či průmyslových systémech je obtížně prosvětlit celou vrstvu kultivačního média, což vede k tvorbě tmavých zón, v kterých nejsou mikrořasy dostatečně osvětlovány. V důsledku nedostatečného míchání může také docházet k nerovnoměrnému využívání živin obsažených v kultivačním médiu nebo může docházet ke vzniku teplotních gradientů. Častým problémem je při zvětšování měřítka systému také tvorba biofilmu na transparentních plochách fotobioreaktorů, což dále snižuje intenzitu působícího světelného záření. Všechny tyto provozní parametry jsou výrazně ovlivňovány hydrodynamickými podmínkami v kultivačních systémech. Studium hydrodynamických podmínek je důležitá zejména při zvětšování měřítka kultivačních systémů. Efektivní promíchávání kultivačního média může: 1) umožnit, aby se všechny buňky mikrořas dostaly do ozařovaného prostoru (světlá zóna) kultivačního média; 2) zabránit vzniku teplotních gradientů nebo sedimentaci buněk mikrořas; 3) zintenzivnit přenos hmoty, což vede k účinnějšímu využívání živin. Intenzivnější promíchávání v blízkosti transparentních ploch může dále zvýšit lokální hodnoty smykového napětí, které ovlivňuje tvorbu biofilmu. Cílem této práce bylo studium vlivu hydrodynamických podmínek na parametry ovlivňující produkci mikrořas ve dvou kultivačních systémech: hybridní horizontální trubkový fotobioreaktor a uzavřený deskový fotobioreaktor. Za tímto účelem byl vytvořen multifyzikální model, který umožňuje detailně studovat vliv hydrodynamických podmínek na proces kultivace mikrořas. Na základě studia stávajících konstrukcí fotobioreaktorů byla navržena řešení pro zintenzivnění míchání kultivačního média s cílem zajistit homogenitu hydrodynamických podmínek v celém objemu zpracovávaného kultivačního média a zvýšit tak produkci mikrořas. Na základě experimentálního měření byl validován numerický model simulující hydrodynamické podmínky v transparentních trubkách hybridního trubkového fotobioreaktoru. Pomocí modelu trasování pohybu částic bylo možné simulovat pohyb buněk mikrořas pro různé provozní konfigurace. Pomocí vytvořeného modelu byl zkoumán vliv různých provozních podmínek na míchání kultivačního média. Při navýšení průtoku kultivačního média byly eliminovány zóny v zadržovacích tancích, kde médium proudí nízkou rychlostí a mohli by zde docházet k sedimentaci buněk mikrořas. Model ukázal, že smykové napětí, které je důležité zejména z hlediska tvorby biofilmu, se zvyšuje v závislosti na rostoucí rychlosti proudění. Výsledky distribuce částic byly integrovány do multifyzikálního modelu, který dokáže predikovat vliv hydrodynamických podmínek na proces kultivace mikrořas. Vytvořený multifyzikální model tak umožňuje zkoumat vliv provozních podmínek na distribuci světla v kultivačním médiu a produkci mikrořas. Model byl kalibrován a validován na základě údaje ze dvou intenzivních experimentálních kampaní. Multifyzikální model zohledňuje vliv vzdálenosti proudících buněk mikrořas od ozařované stěny fotobioreaktoru, která je výrazně ovlivňována promícháváním kultivačního média. Model ukázal, že v systémech pracujících s velkou vrstvou kultivačního média nebo s vysokou koncentrací mikrořas zvyšuje intenzifikace míchání celkovou produkci mikrořas. Z tohoto důvodu je potřeba hydrodynamické podmínky kultivačního média co nejvíce přiblížit stavu, kdy je celý objem média ideálně promícháván. Tohoto stavu lze v případě trubkového fotobioreaktoru dosáhnout zvýšením průtoku nebo použitím statických směšovačů, které by byly instalovány v transparentních trubkách. Hydrodynamické podmínky v deskovém fotobioreaktoru jsou v porovnání s hybridním trubkovým fotobioreaktorem výrazně komplikovanější. Změnou konfigurace nátoku a odtoku z komory deskového fotobioreaktoru je možné sledovat vliv geometrie a provozních podmínek na hydrodynamiku tohoto systému. Porovnáním vytvořeného hydrodynamického modelu s experimentálními měřeními bylo možné zkoumat vliv hydrodynamiky na prevenci tvorby biofilmu. Vytvořený hydrodynamický model dále umožnil optimalizovat provozní a konstrukční parametry deskového fotobioreaktoru. Výsledky ukázaly, že pouhou změnou konfigurace vstupního a výstupního hrdla nebo změnou průtoku kultivačního média nelze zcela eliminovat tvorbu mrtvých zón v komoře deskového fotobioreaktoru. Za účelem eliminace těchto zón byl navržen statický směšovač, který byl instalován v komoře fotobioreaktoru. Numerický model hydrodynamických podmínek v komoře se statickým směšovačem byl validován na základě experimentálních měření. Ve srovnání s prázdnou komorou deskového fotobioreaktoru se při použití statického směšovače zkrátila doba homogenizace a proudění média bylo rovnoměrně distribuováno po celém průřezu komory. Na základě experimentálních měření a numerických modelů byly optimalizovány provozní podmínky v hybridním horizontálním trubkovém fotobioreaktoru a deskovém fotobioreaktoru. Pro další zintenzivnění míchání a homogenizaci proudění kultivačního média byl navržen statický směšovač, který prokázal pozitivní vliv na hydrodynamické podmínky kultivačního systému. Vytvořený numerický model prokázal, že je užitečný nástroj využitelný k optimalizaci stávajících kultivačních systémů, k studiu provozních parametrů při zvětšování měřítka kultivačních systémů nebo pro návrh nových fotobioreaktorů.
Enginyeria ambiental

Les microalgues sont des organismes photosynthétiques considérés pour la production d'énergie. Les photobioréacteurs sont des systèmes fermés avec des productivités plus importantes que les bassins ouverts. Cette étude concerne l'expérimentation et la modélisation d'un réacteur du type « airlift à circulation interne». Des microparticules sont ajoutées pour modéliser les concentrations d'algues dans le réacteur. Les fractions du gaz et les vitesses du liquide ne sont pas affectées par la présence de microparticules. La distribution de la lumière dans le réacteur est calculée en considérant les concentrations de la biomasse et les propriétés optiques des algues. La lumière est atténuée de la paroi jusqu'au centre du réacteur et cette atténuation augmente avec la concentration de la biomasse. Les productivités de biomasse, estimées en utilisant deux modèles biologiques, montrent que celles obtenues dans les photobioréacteurs sont plus élevées que celles obtenues dans les bassins ouverts. La capacité du réacteur pour absorber du CO2 et produire de l'O2 est évaluée selon les productivités obtenues. A intensités lumineuses modérées, l'O2 dissout n'atteint pas le niveau d'intoxication, même pour de faibles débits d'air. Le CO2 et le CIT deviennent limitant si de l'air naturel est injecté dans le bioréacteur. Il est donc nécessaire d'injecter de l'air enrichi en CO2. L'hydrodynamique d'un réacteur airlift hélicoïdal est également présentée. Deux diamètres de tuyaux sont testés dans la section hélicoïdale. Une corrélation est proposée pour estimer le coefficient de frottement en fonction du Reynolds et le rapport entre le diamètre d'enroulement et le diamètre de tuyau. Pour réaliser la culture de microalgues à l'échelle industrielle, il est indispensable d'obtenir un bilan énergétique positif et de faibles émissions de GES. Pour cela, il faudra intégrer la culture dans des procédés de transformation tels que la production de biodiesel et la digestion anaérobique
Microalgae are photosynthetic organisms considered today for energy production. Photobioreactors are closed systems that present higher productivities than open ponds. In this study, a hydrodynamic model is developed for an internal airlift reactor and validated experimentally. Microparticles are added to the reactor at concentrations found in current microalgae cultures. Results show that gas hold-up and liquid velocities are not affected by the presence of particles. Light distribution and availability in the internal airlift reactor is calculated, taking into account biomass concentrations and algae optical properties. Light is attenuated from the wall to the reactor center while this attenuation increases with biomass concentrations. Based on two biological models, biomass productivities achieved in photobioreactors are higher than in open ponds. From biomass productivities, the reactor capacity to absorb CO2 and to release O2 is estimated. Results show that at moderate irradiances, dissolved O2 levels do not reach intoxication at low air flow rates. If natural air is injected into the reactor, CO2 and TIC become limiting therefore, it is necessary to inject CO2-enriched air. The hydrodynamics of a helical airlift reactor is also presented. Two pipe diameters are tested in the helical section. A mathematical correlation is proposed to estimate the friction factor as a function of the Reynolds number and curvature ratios. To perform microalgae culture at large scale, it is necessary to obtain a positive energy and GHGs balance. Therefore, microalgae culture has to be integrated in a system where conversion processes such as biodiesel production and anaerobic digestion are performed

Microalgae are promising candidates for biofuel production, CO2 biomitigation, and production of a variety of value-added products. However, high production costs and large energy consumption have been a major concern hindering the commercialization of microalgal products and processes. In addition, biological contamination and oxygen stress are two of the major contributors to these challenges. The objective of this project was twofold: 1) developing a novel strategy for control of biological contamination to enable non-sterile cultivation of microalgae such as N. oleoabundans, and 2) developing advanced deoxygenation mechanisms to reduce oxygen accumulation in the culture. It was found that addition of appropriate amount of NaHCO3 could effectively inhibit the growth of protozoa while its inhibition on microalgae was much less and could be alleviated by increasing pH to an appropriate level. It was also found that adding 160 mM NaHCO3 in media or decreasing incident light intensity to 100 W/m2 would help alleviate the oxidative stress to cells at 400% of air saturation. The feasibility of contamination-free non-sterile cultivation of freshwater green alga N. oleoabundans was verified using long-term continuous cultivation in a 15-liter TPBR with non-sterile medium and aeration. Furthermore, localized oxygen removal using hydrophobic hollow membranes was found to effectively reduce dO2 and increase lipid accumulation. These results have the potential to be translated into low-cost cultivation of freshwater microalgae processes for production of value-added microalgal products. At a more fundamental level, the mechanisms of the inhibition of NaHCO3 on microalgae and protozoa were discussed. Efforts were also made to simulate the effects of incident light intensity on light distribution, cell growth kinetics, and lipid accumulation of N. oleoabundans under non- sterile cultivation conditions.

Treatment of waste water by freshwater microalgae is rapidly becoming a popular sewage treatment practice throughout the world, but only a few studies have been made into the use of marine strains or of their potential for heavy metal removal. This study examines the heavy metal tolerance of a range of marine microalgal species and examines some of the biological processes involved in metal uptake. Initially over 350 marine microalgal species/isolates were screened for (a) growth in the presence of heavy metals (10 ppm), (b) growth in the presence of wastewater (1:1 sewage : seawater), and (c) heavy metal uptake. Of the microalgae examined, only one isolate, Tetraselmis sp. (TSAW92) was found to satisfy all of the screening conditions. Metal uptake was found to be a biphasic process, with an initial rapid saturable metabolism-independent stage followed by a slower nonsaturable metabolism-dependent stage. Using a novel filtration technique metal uptake was measured at ten second intervals. The results show that most of the metal uptake occurs within one minute after exposure. The second stage of metal uptake was found to be associated with the extracellular release of up to three copper binding proteins of ca. 28, 30, 55 kDa. Protein release was specifically induced by the presence of heavy metals and was not due to metal mediated increases in cell membrane permeability. Two practical applications of Tetraselmis sp. (TSAW92) metal uptake were investigated. First dried biomass was evaluated in a simple packed column. Second, live cells were grown on a wastewater treatment raceway (2m), upstream of a Dunaliella salina stocked nutrient removal raceway. Dried Tetraselmis sp. (TSAW92) was capable of removing copper from a 1:1 seawater : sewage solution to a final loading of 30% at an efficiency of approaching 100%. Over four weeks live TSAW92 removed 100% of the applied copper to a final loading of 37%, and, after the second stage, the raceway was found to remove 95% of the applied nitrogen and 87% of the applied phosphorus. This thesis shows that marine microalgae are capable of removing heavy metals from wastewater in vitro, on raceways, and as dried biomass. Live marine microalgae on raceways were further found to remove inorganic nutrients (nitrogen and phosphorous). In addition work is presented which suggests that copper uptake by the marine chlorophyte Tetraselmis sp. (TSAW92) is mediated through extracellular copper binding proteins.

Philosophiae Doctor - PhD
Biofuel production from microalgae is currently not economically competitive with fossil fuels due to high operational costs. A sustainable system needs to be developed which considers cultivation, harvesting and conversion to fuels as a single loop. The harvesting step has been identified as a major bottleneck within the biofuel production process, contributing to a significant proportion of the operational cost (20-30%). Chemical flocculation is a more affordable alternative to centrifugation and filtration. Chemical flocculants however negatively impact the quality of biomass and conversion efficiency to biofuel by increasing biomass ash content. Bioflocculation with biopolymers or microbes have a minimal impact on the quality of biomass. In this study, the interaction between the filamentous fungus Isaria fumosorosea and the microalgae C. sorokiniana is investigated. Under strict autotrophic conditions at pH 7-8, co-culture of microalgae (2-20 μm) with fungal blastospores resulted in theidevelopment of large pellets (1-2 mm) which may be easily harvested by sedimentation or filtration at 95% harvesting efficiency. Fungal assisted bioflocculation was compared to other harvesting methods with respect to cost and impact on the hydrothermal conversion process. Low cost carbon sources, including waste hydrothermal nutrients, minimal sugar concentrations and algal exudate may reduce fungal cultivation costs. Waste products, such as organic carbon, N, P, CO₂ and trace metals can be recycled and used for algae and fungal cultivation, closing the loop to make the system sustainable.
National Research Foundation; Swiss Government

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Global energy needs are steadily increasing, driven by the rise in both industrialization and population. The major sources of energy are non-renewable and highly polluting. Therefore, the demand for renewable fuels is increasing. One source of renewable fuels is biomass, such as that of microalgae. Microalgae biomasses are a source of lipids and antioxidant pigments and have great potential for biodiesel production and lutein, which have high commercial value. Therefore, in this study I tested the feasibility of producing biodiesel and lutein from microalgae of extremophile environmental of Central Brazil. The freshwater microalgae T. minimum, P.morum, D. communis, N. digitus, E. planctonicus and A. obliquus were collected, isolated, and cultivated in the laboratory, comparing their lipid productivity with that of soybean and lutein productivity with that of Marigold, which is the main raw material for this pigment . The percentage of Fatty Acid Methyl Esters (FAME) of microalgae varied from 9.6 to 21.3%, being A. obliquus the species with the highest content of fatty acids, E. planctonicus with the highest content of lutein and D. communis the one with the highest productivity of biomass, biodiesel and lutein. The D. communis fatty acid profile was also suitable for biodiesel production, since it produced up to 21 times more biodiesel than soybean, but consuming up to 4.8% of the area and 42 times less water. Microalgae cultivation for biodiesel production can be done with less complex and low-cost culture media, including waste water from biodiesel plants. Thus, although the BBM medium had higher biodiesel productivity, the less nutritive media (DAF + OGR and DAF) also yielded adequate biodiesel amounts, being 72% cheaper than BBM. Thus, the biodiesel yield of A. obliquus grown in DAF + OGR was 46 times higher than that of soybeans, using 7.2% of water and 2.2% of the area. These results demonstrate the potential of microalgae in the biodiesel production chain
A demanda por energia aumenta continuamente em nível mundial, impulsionadas tanto pelo aumento da industrialização como também pelo aumento populacional, sendo grande parte dessas necessidades ainda supridas fontes não renovável e altamente poluente e, portanto, limitados às reservas existentes. Assim, a procura por combustíveis derivados de fontes renováveis vêm aumentando a cada dia, sendo intensa a busca por biomassa tais como as microalgas, as quais, por serem fontes de lipídios e pigmentos antioxidantes, vêm sendo estudadas com respeito a seu potencial para produção de biodiesel e luteína, que possui alto valor comercial. Dessa forma, este estudo buscou avaliar a viabilidade da utilização de microalgas de ambientes extremófilos do Brasil Central na produção de biodiesel e luteína. As microalgas dulcícolas T. minimum, P.morum, D. communis, N. digitus, E. planctonicus e A. obliquus foram coletadas em ambientes extremófilos, isoladas e cultivadas em laboratório, tendo sua produtividade lipídica comparada com a da semente de soja e a produtividade de luteína comparada com as flores de Marigold, que é a principal matéria-prima para tal pigmento. As microalgas extremófilas apresentaram potencial para biodiesel, gerando porcentagem de ésteres metílicos de ácidos graxos variando de 9,6 a 21,3%, sendo A. obliquus com maior teor de ácidos graxos, E. planctonicus com maior teor de luteína e D. communis com maior destaque na produtividade de biomassa, biodiesel e luteína. D. communis também apresentou perfil de ácidos graxos adequados para biodiesel, além de produzir até 21 vezes mais biodiesel que a semente de soja. Através das estimativas comparativas concluiu-se que D. communis consumiria até 4,8% da área e 42 vezes menos água do que a usada pelo cultivo de soja para produzir a mesma quantidade de biodiesel. Verificou-se que o cultivo de microalgas para a produção de biodiesel pode ser feito com o uso de meios de menor complexidade e custo, usando inclusive águas residuais que podem ser encontradas em usinas de biodiesel. Assim, apesar do meio de cultivo convencional BBM apresentar maior produtividade em biodiesel, os meios com menos nutrientes (DAF+OGR e DAF), também proporcionaram adequados rendimentos em biodiesel, sendo 72% mais econômicos que o meio BBM. Assim, verificou-se que para A. obliquus cultivada no meio de menor custo, DAF+OGR, a produtividade em biodiesel foi 46 vezes superior ao da soja, com uso de 7,2% de água e 2,2% da área usada no cultivo de soja para produção de mesma quantidade de biodiesel, mostrando o potencial das microalgas para a cadeia de produção e uso de biodiesel.

Les microalgues sont des microorganismes photosynthétiques avec un grand potentiel industriel. Néanmoins, contrôler les conditions optimales de cultures algales à grande échelle reste un défi difficile. L'objectif de cette thèse est de mieux comprendre ces systèmes à l'aide de modèles mathématiques basés sur des équations différentielles. Ces modèles décrivent la croissance microalgale dans des cultures à haute densité en fonction de différents facteurs comme la disponibilité en lumière et en nutriments. Dans une première partie, on étudie l'impact de la photoinhibition et de la turbidité du milieu sur la croissance microalgale limitée par la lumière. Ensuite, le comportement à long terme d'une population microalgale colimitée par un nutriment et la lumière est analysé. Des conditions pour éviter l'extinction de la population sont identifiées. En particulier, on montre que le fonctionnement en continu, avec entrées périodiques (concentrations dans l'alimentation et taux de dilution périodiques) sous variations périodiques de l'environnement (source de lumière et température), conduit la population à un état périodique. Dans une troisième partie, une stratégie est proposée pour maximiser la productivité microalgale en extérieur, en contrôlant l'ombrage. Par la suite, dans le contexte du traitement des eaux usées, on détermine numériquement la profondeur optimale pour une culture limitée par la lumière et par un substrat (à dépolluer). Dans une dernière partie, un modèle est proposé et validé pour rendre compte de la limitation par la lumière, l'azote et le phosphore, tout en incluant la dynamique de photoacclimatation
Microalgae are photosynthetic microorganisms with a high biotechnological potential. They have many industrial applications, including biofuel and wastewater treatment. Nevertheless, controlling optimal growth conditions for full-scale outdoor cultivation of microalgae is challenging. Mathematical models based on differential equations are of great help to better manage these nonlinear and dynamical systems. The aim of this thesis is to better understand how different factors such as the availability of light and nutrients affect microalgae growth in high density cultures. In a first part, we study the impacts of photo-inhibition and medium turbidity when microalgae growth is only limited by light. Then, we analyse the long-term behaviour of a microalgae population accounting both for nutrient and light limitations. We determine the conditions to avoid population extinction. In particular, we show that continuous periodic culture operation (periodic dilution rate and nutrient supply) under periodic fluctuations of environmental conditions (such as the light source or temperature) leads to a periodic behavior. In a third part, we show how to maximize microalgae productivity. We determine a strategy for shading outdoor cultures to protect microalgae from excess light. We also find the optimal incident light for photobioreactors operated at steady state. In the context of wastewater treatment, we determine numerically the optimal depth of a culture limited by light and nutrient. Finally, the last part of this work proposes and validates a mathematical model accounting for light, nitrogen, and phosphorus limitations, including photoacclimation dynamics

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Aquicultura, Florianópolis, 2010
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Dentre as numerosas aplicações comerciais do uso de microalgas podemos destacar sua importância na aqüicultura onde podem ser empregadas como fonte primária de alimento para larvas, organismos jovens e até de adultos de moluscos, camarões e peixes marinhos, uma vez que, o elevado valor nutricional das microalgas justifica sua necessidade na alimentação destes organismos. Muitos fatores influenciam o crescimento das microalgas, sendo que, a disponibilidade de nutrientes é bastante relevante nesse aspecto. Para determinar o crescimento das culturas das microalgas Thalassiosira weissflogii, Chaetoceros muelleri, Isochrysis sp. e Pavlova sp. quando cultivadas com diferentes concentrações de nitrogênio e fósforo durante todo o cultivo, foram desenvolvidos cultivos experimentais com essas espécies. As espécies foram cultivadas em meio de cultura Guillard (1975) e a diferença dos tratamentos foi a quantidade de nitrogênio e fósforo utilizada nas soluções estoques para compor o meio de cultivo. Os parâmetros de crescimento analisados foram a densidade celular máxima, o tempo de cultivo e a velocidade de crescimento. Foi aplicado um delineamento uni-fatotorial para blocos casualizados.Os dados obtidos foram submetidos à analise de variância fatorial (Anova com P<0,05) e quando necessário ao Teste de Comparação de Médias de TuKey (p<0,05). Quanto ao crescimento das culturas o emprego do meio f - com o dobro da concentração de nitrato de sódio e de fosfato de sódio descrita em Guillard (1975), promoveu uma maior densidade celular nas culturas de Thalassiosira weissflogii, (66,7 x 104 cel/mL) Chaetoceros muelleri, (1566,7 x 104 cel/mL) Isocrysis sp (1903,3 x 104 cel/mL) e Pavlova sp (2362,3 x 104 cel/mL). (p<0,05) Quanto ao tempo de cultivo e a velocidade de crescimento não houve diferença significativa para nenhuma das espécies (p<0,05).

The environmental conditions created in the Southwestern deserts of the United States are conducive to the production of green microalgae biomass, for use as a feedstock intended for conversion to carbon neutral liquid biodiesel. One promising heat-tolerant, rapidly-growing, high lipid content species is the chlorophyte, Chlorella sorokiniana (Shihira and Krauss, 1965) (isolate DOE 1412), which has been selected for pilot-scale production as part of a larger algal biofuels project to assess its potential for long-term productivity in open, outdoor monoculture production systems. Molecular analysis exposed the presence of the pathogenic bacterium, Vampirovibrio chlorellavorus (Gromov & Mamkaeva, 1972) causing infection and death of DOE 1412, which occurred most rapidly at air temperatures exceeding 34 °C. This Gram-negative bacterium has been reported to attach to and utilize the cellular contents of several Chlorella species, leading to yellowing and flocculation of algal cells, and death of the host. A quantitative PCR assay was developed to monitor pathogen accumulation using the 16S ribosomal RNA gene, in addition to the algal 18S ribosomal RNA gene for normalization. The assay is highly sensitive, with limits of quantification for the 16S and 18S gene targets calculated to be 19 and 131 copies, respectively. The qPCR assay was used to monitor several outdoor reactors inoculated with the DOE1412, throughout the warm season growth-to-harvest cycle to understand the disease cycle and inform disease management decisions. Further, the bacterium was monitored in paddlewheel DOE 1412 cultures treated with benzalkonium chloride (BAC), a biocide tested for the ability V. chlorellavorus attack of DOE 1412. The treatment resulted in a reduced growth rate for DOE 1412, but prolonged the duration of the production cycle resulting in increased total harvestable yield, compared to untreated control cultures.

SILVA, Jose William Alves da. Cultivo da microalga Chlorella vulgaris em efluentes aquícolas e sua influência na concentração lipídica. 2013. 48 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2013
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Microalgae can be used as feedstock for producing biofuels on a large scale, due to the ease of cultivation, strong growth rate, high content of fatty acids and higher productivity than other oils, making it an excellent alternative to fossil fuels. The study evaluated the use of microalgae Chlorella vulgaris in phytoremediation of aquaculture effluents and lipid evaluation. The microalgae was obtained in the algae bank Federal University of Ceará. Were three treatments with six replicates, using as culture medium Guillard f / 2 and effluents of shrimp and fish farming. Cultivation was batch containers of 12 L and was observed daily by spectrophotometry at 680 nm, and determining the concentration of ammonia, nitrite, nitrate and phosphorus performed at the beginning, middle and end of crops. The separation of the cells from culture medium was performed by chemical flocculation using NaOH 2N. After washing, the biomass was dried in an oven with renovation of air at 60 °C for 24 hours and then quantified. Trea tment with effluent was, as biomass production, significantly higher than the others, showing an average weight of 0.91 ± 0.05 g L-1 . The effluent removed satisfactorily nitrogen compounds with a 76% removal of these compounds. The cultivation performed with tilapia effluent showed better lipid productivity with 0.025 ± 0.002 g L -1 day -1. The microalga C. vulgaris can be used for phytoremediation of aquaculture effluent, biomass and lipids.
As microalgas podem ser utilizadas como matéria prima para a produção de biocombustíveis em larga escala, em decorrência da facilidade de cultivo, acentuada velocidade de crescimento, alto teor de ácidos graxos e produtividade maior que outras oleaginosas, sendo uma excelente alternativa aos combustíveis fósseis. O experimento avaliou a utilização da microalga Chlorella vulgaris na fitorremediação de efluentes aquícolas e sua produção lipídica. Foram realizados três tratamentos, com seis repetições cada, utilizando como meios de cultura o Guillard f/2 e efluentes da carcinicultura e piscicultura. O cultivo estacionário foi realizado em um recipiente com volume útil de 12 L e foi acompanhado, diariamente, por espectrofotometria a 680 nm, sendo a determinação das concentrações de amônia, nitritos, nitratos e fosfatos realizada no início, meio e fim dos cultivos. A separação das células do meio de cultivo foi realizada por floculação química, usando NaOH 2N e, depois de lavada, a biomassa foi seca em estufa com renovação de ar a 60 °C por 24 h. O tratamento com efluente da carcinicultura apresentou uma produção de biomassa significativamente maior que os demais, com valor médio de 0,91±0,05 g L-1. Os compostos nitrogenados presentes nos efluentes atingiram remoção média de 76%, resultado considerado satisfatório. O cultivo realizado com efluente de piscicultura apresentou melhor produtividade lipídica com 0,025 ± 0,002 g L-1 dia-1. Assim, a microalga C. vulgaris pode ser utilizada na fitorremediação de efluentes aquícolas para a produção de biomassa e extração de lipídios.

L‘objectif général du présent travail est la conception et le développement d‘un procédé original de culture de microalgues pour la production à grande échelle d‘huiles végétales à faible coût pour le marché du biodiesel. Une procédure multicritères de sélection de souches a d‘abord été mise au point afin d‘identifier la souche la plus susceptible de répondre favorablement aux paramètres du procédé préalablement déterminés. Cette procédure permet d‘inclure un grand nombre de critères et de considérer l‘importance relative de chacun de ces critères dans le pointage final accordé aux souches présélectionnées. Elle peut aussi être transposée à d‘autres contextes techniques et géographiques pour la sélection de souches destinées à diverses applications commerciales. Par la suite, l‘évaluation de la croissance et de la productivité lipidique de la souche sélectionnée a été effectuée au laboratoire, en mode nutritionnel mixotrophe, en présence d‘un important coproduit de l‘industrie laitière : le perméat de lactosérum. Le profil lipidique de la souche privilégiée, ainsi que sa capacité à hydrolyser le lactose, mise au jour pour la première fois, ont permis de démontrer le potentiel du procédé. Une méthode de suivi de consommation et d‘hydrolyse du lactose contenu dans le perméat de lactosérum a été développée par l‘intermédiaire de la technologie « spectroscopie infrarouge à transformée de Fourier » couplée à une sonde externe avec « prisme à réflectance totale atténuée » (FT-IR/ATR) ainsi que la conception de modèles univariés et multivariés de type « régression des moindres carrés partiels (PLS) ». Ce type de modèle est plus précis que ceux déjà existants pour le suivi des sucres dissous par la méthode infrarouge car l'utilisation de régressions PLS permet de tenir compte du phénomène d‘additivité des spectres lorsque différents analytes pouvant absorber dans une même région du spectre sont présents dans la solution. De plus la méthode développée permet d‘envisager un suivi in situ de la consommation des sucres dans une culture de microorganismes à l‘échelle industrielle. La méthode des surfaces de réponse (RSM) a ensuite été utilisée afin d‘optimiser les conditions de pH et de densité de biomasse permettant de provoquer une accumulation de lipides à l‘intérieur de cultures de microalgues carencées en azote. Des modèles prédictifs ont été construits pour trois souches différentes et ce en modes nutritifs photoautotrophe et hétérotrophe. Ces travaux ont permis de démontrer l‘existence d‘une interaction entre ces deux facteurs. De plus, certaines différences spécifiques ont pu être mises en évidence en comparant les réponses entre les souches. À la lumière des résultats obtenus, certains paramètres d‘optimisation devront être considérés dans le cadre d‘un procédé de production de biomasse microalgale oléagineuse en deux phases; la première phase permettant d‘obtenir une productivité en biomasse maximale par une stratégie d‘augmentation du taux d‘hydrolyse du lactose; la seconde phase permettant une accumulation de lipides grâce à l‘imposition de conditions environnementales spécifiques. L‘augmentation des connaissances disponibles sur les microalgues ainsi que l‘amélioration et la standardisation des techniques de génie génétique nous permettront d‘aborder ces problématiques d‘un point de vue différent dans le futur.

A Report by The Water Resources Research Center, The University of Arizona, Tucson, Arizona, for The Solar Energy Research Institute, 1617 Cole Boulevard, Golden, Colorado 80401 / Subcontract No. XK-5-05083-1) / June 25, 1986.
Summary: The Arizona Water Resources Research Center was contracted by the Solar Energy Research Institute (SERI) to conduct an inventory of saline water resources within the State of Arizona. The purpose of this inventory was to identify on the basis of availability, chemistry, sustained yield, and institutional factors, the potential saline water supplies sufficient to maintain a microalgae production facility for eventual conversion of the algal biomass to synthetic fuel. Criteria for the inventory were specified by SERI. The six subtasks associated with the project are identified on the attached flow chart. The method of investigation involved a literature and data survey and interviews with key personnel in government or quasi-official agencies. The principal data are included in the report together with a list of contacts. As a result of the survey conducted by the Water Resources Research Center, six focal areas with saline ground-water resources were identified and ranked. The report includes a summary of the selection methodology, a description of the hydrogeology of each site and a thorough discussion of relevant institutional considerations, including permitting requirements under current ground-water associated laws in Arizona. Detailed maps are included of the saline surface water and ground-water sources. Recommendations are included for further evaluation by SERI of areas potentially suitable for a project.

The growing industrialization, the high level of pollution, and the reduction of resources lead to the necessity to find an alternative way to handle the entire production chain from the raw materials to the finished products. The zero-waste strategy and the circular economy are the best solutions to remediate the dramatic conditions in which our planet pours. Moreover, the reduction of fossil fuels reserves and the continuously increasing demand for energy around the world has led to the necessity to find an eco-sustainable alternative to conventional fuels. In the last few years, biofuel production from different plant sources has been increasingly studied by researchers. The production of third-generation biofuels from raw materials that do not compete with food crops is attracting more and more attention. Third-generation biofuels can be produced from microalgal biomasses or from their intracellular components such as lipids. Moreover, their production, if compared to conventional biomasses, reduces land and water utilization along with the use of pesticides. Microalgae are unicellular microorganisms able to grow under autotrophic, heterotrophic or mixotrophic conditions depending on the carbon source used in their metabolism as well as light conditions. They are composed mainly of lipids, proteins and carbohydrates, whose relative proportions depend in particular on the species and growth conditions. They are generally used for human or animal nutrition, or extraction of added-values components for chemical and pharmaceutical industries, but also for biofuel production. In recent years, lipid, protein, and pigment extraction from microalgae has been widely studied for various applications such as the productions of biodiesel from the lipid fraction and of nutraceuticals and dyes from vitamins, proteins, and pigments. However, it is important to stress that the microalgae biorefinery concept is the only way to make microalgae competitive with products obtained from conventional sources, and that the use of microalgae to produce only biofuels or only nutraceuticals has not yet reached clear-cut economic feasibility. The biggest challenges are the relatively high cost of biomass production and the energy demand for the extraction and separation processes. Therefore, in order to make microalgae products economically viable and increase their marketability, it is necessary to reduce costs, for example by valorizing process residues as co-products. It is generally accepted that the sale of co-products will make the production of biofuels from microalgae economically feasible. Indeed, it was estimated that the residue of microalgal biomass after lipid extraction could be worth between 100 and 225 USD per ton and could yield co-products ranging in value from 0.95 to 2.43 USD per gallon of biodiesel produced. Moreover, the microalgae protein fraction has an economic value that ranges from 0.86 USD/kg as feed to 5.57 USD/kg as food. Moreover, thanks to their capability of also metabolizing organic carbon, microalgae can in fact be grown in wastewaters, thus reducing the use of fresh water, the cost of growth medium, the energy consumption and, at the same time, the wastewater polluting impact. There are several studies in the literature focusing on the use of microalgae to treat wastewaters such as municipal and textile wastewaters, among others. In this contest, agri-food wastewaters are good candidates to be used as microalgae medium because they are rich in nutrients and the resulting biomass obtained after treatment could be used for the extraction of high added value components, such as protein and pigments. Between them, winery wastewaters (WWWs) which are released from different activities of the wine making process, namely tank washing, transfer, bottling and filtration, are suitable to be treated by microalgae. The polluting impact of WWWs is related to their high organic load (polyphenolic compounds, sugars, organic acids and esters), low pH (3–5), high content of suspended particles and large volumes (0.5–14 L per liter of wine produced). Owing to the release of organic compounds and inorganic ions, their disposal in land without adequate treatment can change the physicochemical properties of groundwater such as color, pH and electrical conductivity, among others. Whit regard to the open issues recalled above, the research project has aimed to develop a biorefinery from microalgae. Winery wastewaters were used as growth medium both for microalgal biomass production and reduction of pollutant impact, the protein fraction was extracted from the biomass as high added-value component, and the residual biomass was submitted to pyrolysis process to produce biofuels. The topic addressed by this thesis is organized and subdivided into chapters as follows. Chapter 1- Literature review on the biorefinery concepts, the application to microalgae production and the microalgae world situation concerning their metabolisms, growth system, industrial application (wastewaters treatment, extraction of high-added-value components and, biofuels production). Chapter 2- The optimization of winery wastewaters concentration in microalgae growth medium to obtain both high microalgae concentration and productivity and good results in terms of reduction of pollutant impact. Three different winery wastewaters collected from different steps of the winemaking process were studied. The co-culture of Chlorella vulgaris and Arthrospira platensis was grown under continuous light and air supply. The optimized parameters obtained in the previous section were studied at different light conditions to identify the prevalent metabolism of the microalgae to consume the pollutant molecules present in the wastewaters. Moreover, the microalgae growth was performed into different photobioreactor configurations: tubular photobioreactor (TP), column photobioreactor (CP) and, open pond (OP) to improve the biomass concentration and the pollutant impact removal efficiency. Chapter 3- To perform a scale-up of the process the co-culture was grown in 20 L column photobioreactor, the growth medium under the condition optimized in the previous chapters was supplied continuously by a pump system. Chapter 4- The extraction of high-added value components from microalgal biomass were investigated taking into account the biorefinery concept. The optimization of the protein extraction process from A. platensis by Ultrasound-Assisted Extraction was performed using Box-Behnken Design in which the effects of extraction time, solvent volume, and mass of A. platensis were investigated. Moreover, the extraction and purification of c-phycocyanin from A. platensis and the subsequent protein extraction on wet c-phycocyanin residue was performed. The protein extraction from the co-culture grown in the different photobioreactor configurations was carried out under the condition optimized in the previous chapter. Moreover, the effect on cell size and cell wall thickness of growing the co-culture in presence of winery wastewaters was investigated. Variation of protein expression as function of photobioreactor configuration was also assessed. Chapter 5- The energetical recovery of the produced co-culture biomass was investigated. The pyrolysis process was carried out on microalgal biomass obtained from wastewater treatment in membrane photobioreactor. The operational condition of the process and, the distribution of reaction products and their composition ware studied. Chapter 6- A final discussion of the process was performed based on the results obtained from the previous chapters.

Les microalgues sont bien connues pour produire de nombreuses molécules bioactives qui sont de plus en plus utilisées dans les industries pharmaceutiques et agroalimentaires. Mon projet de thèse avait pour but de mettre en évidence des activités antimicrobiennes d’origine naturelle valorisables en cosmétique notamment en tant que conservateurs et antiacnéens. Des milieux de culture en phase stationnaire de croissance d’une collection de 113 microalgues cultivées au sein du laboratoire ont été prélevés. La présence d’activités antibactérienne et / ou antifongique a été évaluée sur différents microorganismes modèles. Ce criblage m’a permis d’isoler 5 microalgues sécrétant des molécules inhibant la croissance de bactéries appartenant aux genres Salmonella, Staphylococcus et Propionibacterium. La suite de mon travail a porté sur la microalgue référencée S555 qui montre une activité d’inhibiton totale de la croissance de 3 bactéries Gram + : S. aureus, S. epidermis et P. acnes, ces 2 dernières espèces étant impliquées dans l’acné. Cette microalgue a alors été mise en culture en pilote pré-industriel afin de confirmer ces inhibitions. Un élargissement du spectre d’action suggère que ces activités sont spécifiques des bactéries Gram positives. De plus, le(s) composé(s) actif(s) de S555 ne présente(nt) aucune cytotoxicité ou potentiel irritant sur des fibroblastes en culture, les rendant potentiellement utilisables en dermocosmétique. Enfin, un fractionnement du milieu de culture et de la biomasse de cette microalgue a été mis en oeuvre pour séparer le(s) composé(s) actif(s). Leur caractérisation est actuellement en cours
Microalgae are well-known to produce many bioactive molecules which are used more and more in both pharmaceutical and agroalimentary companies. The aim of my PhD project was to discover antimicrobial activitiesof natural origin which may be used in cosmetic in particular as preservatives and antiacne. Culture media in stationary phase of growth of a 113 microalgae collection, cultivated in the laboratory, were harvested. The presence of antibacterial and / or antifungal activities was evaluated on different microorganisms. This screening allowed to isolate 5 microalgae secreting molecules inhibiting the growth of bacteria belonging to Salmonella, Staphylococcus and Propionibacterium genera. Then my work concerned the S555 microalgae which show a total inhibiton activity of the growth on 3 Gram + bacteria : S. aureus, S. epidermis and P. acnes, these 2 last species being involved in the acne disease. This microalgae was then cultivated in industrial condition in order to confirm these inhibitions. A widening of the action spectrum suggests that these activities are specific to Gram + bacteria. Moreover, the S555 active compound(s) present no cytotoxicity or irritability potential on fibroblasts in vitro,making them potentially usable in dermocosmetic. Lastly, a fractionation of the culture medium and biomass of S555 microalgae were performed to separate the active compound(s). Their characterization is currently in progress

Cette thèse porte sur la commande d'un photobioréacteur continu pour la culture des microalgues. La modélisation du système et l'identification des paramètres du modèle ont été particulièrement abordées. Les paramètres cinétiques et de transfert de l'énergie lumineuse ont été obtenus à partir des données acquises le long de la phase exponentielle de la culture de Porphyridium purpureum en mode batch sous différentes conditions expérimentales dans un photobioréacteur de 2,5 litres. La concentration en biomasse est la quantité physique la plus importante à réguler. Puisque elle n'est pas toujours facile à mesurer en ligne, un capteur logiciel permettant d'estimer la quantité de biomasse produite à partir des mesures en ligne est proposé. Il s'agit d'un filtre de Kalman étendu (EKF) qui combine le modèle du système avec la mesure de la concentration du carbone inorganique total et d'autres variables physiques (pH, intensité lumineuse,...) afin d'estimer en ligne la concentration de la biomasse. Ce capteur logiciel a été validé expérimentalement en utilisant un nouveau photobioréacteur de 9,6 litres de type colonne à bulles, équipé de plusieurs dispositifs destinés à mettre en place un système de commande et de régulation, et de techniques de mesure permettant d'accéder à l'évolution des principales variables. Plusieurs démarches pour la commande de la culture de microalgues ont été également proposées : commande non-linéaire et linéaire, régulation par PID, commande par modèle générique GMC, commande linéarisante par retour d'état et commande prédictive non-linéaire. Ces différentes commandes ont été mises oeuvre en simulation ce qui nous a permis de choisir les stratégies les plus performantes et de les valider sur le photobioréacteur instrumenté.

Cette thèse porte sur la commande d’un photobioréacteur continu pour la culture des microalgues. La modélisation du système et l’identification des paramètres du modèle ont été particulièrement abordées. Les paramètres cinétiques et de transfert de l’énergie lumineuse ont été obtenus à partir des données acquises le long de la phase exponentielle de la culture de Porphyridium purpureum en mode batch sous différentes conditions expérimentales dans un photobioréacteur de 2,5 litres. La concentration en biomasse est la quantité physique la plus importante à réguler. Puisque elle n’est pas toujours facile à mesurer en ligne, un capteur logiciel permettant d’estimer la quantité de biomasse produite à partir des mesures en ligne est proposé. Il s’agit d’un filtre de Kalman étendu (EKF) qui combine le modèle du système avec la mesure de la concentration du carbone inorganique total et d’autres variables physiques (pH, intensité lumineuse,…) afin d’estimer en ligne la concentration de la biomasse. Ce capteur logiciel a été validé expérimentalement en utilisant un nouveau photobioréacteur de 9,6 litres de type colonne à bulles, équipé de plusieurs dispositifs destinés à mettre en place un système de commande et de régulation, et de techniques de mesure permettant d’accéder à l’évolution des principales variables. Plusieurs démarches pour la commande de la culture de microalgues ont été également proposées : commande non-linéaire et linéaire, régulation par PID, commande par modèle générique GMC, commande linéarisante par retour d’état et commande prédictive non-linéaire. Ces différentes commandes ont été mises oeuvre en simulation ce qui nous a permis de choisir les stratégies les plus performantes et de les valider sur le photobioréacteur instrumenté
This thesis deals with the control of closed microalgal photobioreactor. Mathematical modeling and model parameters identification are particularly approached. The kinetics and light transfer model parameters were obtained by fitting the experimental data acquired along the exponential phase of batch cultivation of Porphyridium purpureum under different conditions in the photobioreactor (2.5 liters). Biomass is one of the most valuable variables to control. Because biomass concentration is very difficult to measure on-line, a software sensor for its estimation is proposed. An extended Kalman filter (EKF) is developed, based on a dynamic process model in combination with total inorganic carbon measurement within an algorithm of state observation. This software sensor leads to good results and was experimentally validated in a new bubble column photobioreactor (9.6 liters) which carried different probes and sensors necessary to implement a control system. Several control strategies for microalgae culture were also proposed: non-linear and linear controllers, PID controller, generic model controller (GMC), input-output linerarizing controller and non-linear predictive controller. We illustrated our approaches with numerical simulations which allowed us to choose the most successful strategies and to validate them on the instrumented photobioreactor

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia Ambiental
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Uma das alternativas de produção de biodiesel, atualmente, em pauta, é o uso de biomassa de microalgas para a obtenção de lipídios, entretanto, existem alguns processos da sua produção que precisam de mais estudos, tais como os insumos utilizados. Uma das alternativas de matéria-prima para o cultivo é o efluente do cultivo superintensivo de camarões marinhos com bioflocos (BF). Dessa forma, o objetivo geral deste trabalho foi realizar a avaliação técnica e ambiental de diferentes culturas para a produção de microalgas destinadas a biodiesel. Para a avaliação técnica, foi considerada a produção de microalgas utilizando três meios de cultivo: (i) f/2 - elaborado com fertilizantes químicos; (ii) BF - emprego de 100% de efluente e (iii) 50/50 - elaborado com 50% de cada. A produtividade foi avaliada em relação a ganho de biomassa e a acumulação de lipídios e ésteres. Para o desempenho ambiental, foi utilizada a ACV, sendo estabelecida a unidade funcional de 1 kg de biomassa de algas contendo 25% de lipídios, produzida em 30 dias. Os limites do sistema vão da aquisição de matéria-prima até a biomassa seca de algas. Foram propostos cenários de avaliação ambiental semelhantes aos anteriores, sendo BF dividido de acordo com o procedimento de alocação adotado: BFa e BFb. Para Avaliação de Impacto Ambiental, foram consideradas categorias do método CML 2000 e a Demanda Acumulada de Energia. A produtividade de biomassa obtida em BF foi 17,5% superior às demais e, em relação à acumulação de lipídios, os valores foram semelhantes em todos os meios. Porém, a produtividade obtida nesses cultivos foi baixa. O consumo de energia foi o principal responsável também pelos principais impactos ambientais observados, no que se refere à acidificação, toxicidade humana e potencial de aquecimento global, sendo BFb o cenário menos impactante e f/2 o mais impactante. eutrofização, o cenário BFa foi o mais impactante devido as emissões de nitrato e fosfato e o cenário BFb apresentou impactos positivos, devido a dispensa do tratamento do efluente do cultivo de camarões. Percebe-se que o sistema de produção necessita de modificações a fim de tornar-se viável em escala industrial, especialmente no que se refere ao consumo de energia elétrica.

Dans l'optique de mieux cerner le mécanisme moléculaire des désaturases, une étude consistant à incuber des cellules entières en présence d'analogues soufrés d'acides gras a été menée sur la microalgue verte Chlorella sorokiniana. L'analyse de la régiocryptochimie des D9 et D12-désaturases a permis de mettre en évidence l'implication d'un mécanisme séquentiel. L'utilisation d'analogues soufrés adéquats a également permis de démontrer la présence d'un transfert d'oxygène hautement stéréosélectif du site actif vers l'atome de soufre pour la D12-désaturase. La détermination des configurations absolues des sulfoxydes obtenus a été envisagée par l'intermédiaire de la résonance magnétique nucléaire à l'aide de réactifs de déplacement chimique. La validité de cette approche a été vérifiée sur des sulfoxydes synthétisés sous forme énantiomériquement pure. Enfin, l'aptitude de cellules entières à oxyder des thioéthers prochiraux a été déterminée en terme de rendement et d'énantiosélectivité.

The main objectives of the Ph.D. thesis were: i. to characterize the growth of Coccomyxa melkonianii SCCA 048 for the production of high-value bio-products, such as lipids; ii. to investigate the growth and lipids production kinetics of the microalga, and the optimization of its biomass production as well as its lipids productivity in qualitative-quantitative terms; iii. to identify the best cultivation strategies of the selected strain for enhanced production of biomass and high-value compounds, with viable exploitation in large-scale plants (Raceways) for several biotechnological applications. Coccomyxa melkonianii SCCA 048, belonging to the Sardinian Culture Collection of Algae (SCCA), is a green extremophile microalga found in a mine drainage habitat and characterized by the ability to live in highly selective environments with extreme conditions, such as high concentrations of heavy metals. This strain has been already elected for its rich carotenoids profile. These features make C. melkonianii SCCA 048 a very promising strain for cultivation in economic open ponds and for the production of high-value products if cultivated under a suitable cultivation strategy. The thesis research aimed to optimize the production of biomass and to study the effect of pH and nitrogen concentration on lipid composition. After the identification and contextualization of the research topic, supported by updated scientific literature, the thesis identifies the effects of the selected parameters on the production of biomass and lipid content in quantitative and qualitative terms. The results identify the optimal pH and nitrogen values for biomass production and highlight the productive potential of the Sardinian microalga C. melkonianii. Furthermore, the research acquired new information on the physiology of this strain, which constitutes a fundamental prerequisite for any future project that foresees the use of the organism on a large scale.

Orientador: Lúcia Helena Sipaúba Tavares
Banca: Odete Rocha
Banca: Maria da Graça Gama Melão
Banca: José Sávio Colares de Melo
Banca: José Augusto Senhorini
Resumo: O objetivo deste trabalho foi analisar a viabilidade da cultura em larga escala e a composição bioquímica da alga Chlorophyceae Ankistrodesmus gracilis e do Cladocera Diaphanosoma birgei em laboratório. O experimento foi realizado no Laboratório de Limnologia e Produção de Plâncton do Centro de Aqüicultura da UNESP (CAUNESP). As algas foram cultivadas em meio Chu12 e NPK (20:5:20), sob iluminação contínua (4.000 e 1.100 lux para pequena e larga escala, respectivamente), aeração constante e temperatura controlada (24,0±2,0ºC). O tempo de produção das algas foi de 31 dias, concentração na fase exponencial variando de 68,50 a 358,38 x 104 células.ml-1 nos diferentes volumes de cultivo. A análise bioquímica revelou que a alga apresenta 32,6% de proteína, 20,0% de carboidrato, 8,0% de extrato etéreo, 18,3% de cinzas, 3,2% de fibras e 4.583 cal/g. O aminograma revelou que 58,7% da proteína era constituída por aminoácidos essenciais. A relação Ca:P foi aproximadamente 1:1. O Cladocera foi oriundo dos viveiros de piscicultura do CAUNESP, isolado e criado em volumes de 500ml a 850L, em sistema estático, alimentados com A. gracilis, em temperatura de 23,3±3,6ºC, fotoperíodo natural e aeração suave. O tempo de produção do Cladocera foi de 27 dias com densidade final de 2.500 indivíduos.L-1. A análise bioquímica deste microcrustáceo revelou uma composição de 70,0% de proteína, 5,7% de carboidrato, 8,7% de extrato etéreo, 11,5% de cinzas, 4,3% de fibras e 4.627 cal/g. Os resultados apresentados sugerem que Ankistrodesmus gracilis e Diaphanosoma birgei são espécies com potencial para cultura e alimentação na aqüicultura
Abstract: The objective of this work was to analyze in detail the viability of the culture and the biochemical composition of the Chlorophyceae algae Ankistrodesmus gracilis and of the Cladocera Diaphanosoma birgei in laboratory. The experiment was carried out in the Limnology Laboratory and Plankton Production in the São Paulo State Aquaculture Center at UNESP (CAUNESP). The algae were cultivated using a culture media of Chu12 and NPK (20:5:20), under continuous light (4000 and 1100 lux for massive and small scale, respectively), constant aeration and controlled temperature (24.0±2.0ºC). Algae production time was of 31 days, concentration during the exponential phase varied from 68.5 to 358.38 x 104 cells.ml-1 in different volumes of culture. Biochemical analyses revealed that the algae presents 32.6% of protein, 20.0% of carbohydrate, 8.0% ethereal extract, 18.3% of ashes, 3.2% of fibers and 4.583 cal/g. The aminogram revealed that 58.7% of the protein was made up of essential aminoacids. The Ca:P relation was of approximately 1:1. The cladoceran was derived from the CAUNESP fish culture ponds, isolated and produced in volumes of 500ml to 850L, in batch system, fed with A. gracilis, at temperatures of 23.3±3.6ºC, natural photoperiod and mild airing. Cladocera production time was of 27 days with final density of 2500 individuals.L-1. The biochemical analyses of this micro-crustacean revealed a composition of 70.0% of protein, 5.7% of carbohydrate, 8.7% of ethereal extract, 11.5% of ashes, 4.3% of fibers and 4627 cal/g. The results here presented suggest that Ankistrodesmus gracilis and Diaphanosoma birgei are species with potential for culture and feeding in aquaculture
Doutor

This is the first study to investigate the nutritional requirements of the penaeid prawn protozoeal phase (the first feeding stage of the prawn life cycle) using micro algae to provide different nutritional conditions. The work was a simultaneous examination of the biochemical composition of the larvae and their microalgal diets. In addition, the influence of naupliar composition on later larval stages was studied. The biochemical parameters measured were total protein, lipid and carbohydrate (i.e. gross biochemical composition) and total lipid was further resolved into individual fatty acids. The nutritional requirements of penaeid prawn larvae were assessed by measuring the survival, development (metamorphosis) and growth (in terms of dry weight) of larvae in response to various algal diets. In this way differences in survival, development and growth could be related to biochemical differences in the algal diets and associated larval body composition. The changes in biochemical composition of *Penaeus japonicus* and *P. monodon* larvae during metamorphosis from nauplii to protozoea 1 (PZl) and the time course of these changes during starvation and feeding for the ~42 h period that the PZl stage lasts, were examined. The larvae utilised lipid as a major energy source during metamorphosis. The fatty acid fraction of the lipid in nauplii was high (60 to 80%) compared with protozoeae (30 to 60%) and provided a large proportion of the energy required for metamorphosis. Of the total fatty acids, the monounsaturated (MUFAs) and saturated fatty acids contributed most of this energy. During starvation the MUFAs and polyunsaturated fatty acids (PUPAs) were metabolised while the highly unsaturated fatty acids (HUFAs) were conserved, presumably because of their role as structural components in cell membranes. The PUFA linoleic acid (18:2n-6) appeared to have a role as a component of cell membranes when in short supply, but it accumulated as an energy reserve when in excess in the diet. Linolenic acid (18:3n-3) was actively metabolised to other membrane fatty acids or used for energy. The effect of altering the biochemical composition of the microalga *Tetraselmis suecica* on *P. semisulcatus* larvae was studied by reducing the nitrate concentration from -1760 μ*M* to 176 μ*M* in the culture media. Carbohydrate increased three fold in the low nitrate algae, and protein and lipid were reduced slightly compared to the control. The low protein:energy ratio (0.1 to 0.2) of the low nitrate diets resulted in a delay in the development of the larvae compared to that of the animals fed the control diet (ratio 0.3 to 0.4). Survival was not affected by the algal diets. Four species of algae (*Tetraselmis suecica*, *Chaetoceros muelleri*, Tahitian *Isochrysis* sp. (T-iso) and *Dunaliella tertiolecta*) differing predominantly in their fatty acid composition were fed to *P. japonicas* larvae as single species diets. The two best diets (in terms of growth and survival of the prawn larvae) were subsequently fed in combination to *P. semisulcatus* and *P. monodon* larvae to assess their nutritional value as part of a mixed diet. The survival and development to mysis 1 (M1), i.e. performance, of the larvae was affected by the algal diets such that the diets could be ranked: *C. muelleri* > *T. suecica* > *Isochrysis* sp. (T-iso) > *D. tertiolecta*. The fatty acid profiles of the algae, particularly those of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-6), were related to those of the larvae and their performance. The presence of both of these fatty acids in the algal diet was necessary to produce high performance whereas docosahexaenoic acid (22:6n-3) was not. The low requirements for the PUPAs 18:2n-6 and 18:3n-3 were modulated by the presence of HUFAs such as 20:4n-6, 20:5n-6 and 22:6n-3. Therefore when these HUFAs were present in the diet, less 18:2n-6 and 18:3n-3 were required. However high proportions of 18:2n-6 and 18:3n-3 alone, did not replace the requirements for 20:4n-6, 20:5n-6 and 22:6n-3. Understanding the nutritional requirements of penaeid prawn larvae will lead to the production of a cost effective and optimum diet for use in hatcheries. In addition, this research will contribute to the production of a purified artificial diet for penaeid prawn larvae which can be used to examine the requirements for other nutrients.

LOPES, Pedro Henrique Martins. 2008. 94f. Dissertação(Mestrado em Engenharia de Pesca)- Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2008.
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Seaweed consists in a vital resource to the economy of many countries and it has been explored all over the world thanks to its capacity of producing colloids such as agar, carrageen and sodium alginate as well as the useful aspect either in food industry, fertilizers or medicine area. Studies on induction, cultivation and callus reorganization have been shown an important issue on tissue culture technique and its usage. In different tissue experience in vitro the usage of regulating growth is so fundamental to establish the competence and necessary conditions to callus formation and regeneration. In this present study has been tested the auxin and citocinin effects in different dosages of agar in ASP 12-NTA. The indolacetic acid effect and 6- benzilalaminopurine have been tested as well in seaweed in the species Gracilaria birdiae with two separated doses, 0,5mg/L and 0,8mg/L and together in concentration of 1:5mg/L and 5;1mg/L. Regarding to agar concentration, it has been tested two concentrations with 0,5% and 0,8% in all treatments. As to sterilization process has been done with fungicide (nistatina) and antibiotic (ciprofloxacin) besides iodopovidona and the sodium hypochlorite. The complete experiment had been lead in a germination chamber with 25±2ºC and photoperiod of more or less 16 hs of light and 08hs dark and 30,2‰ saltiness, pH around 7,6 within 60 days approximately. In more or less 50 days of cultivation had been noticed callus formation and regeneration process therein. The results had been underwent by statistic analyze treatments of contingent through (x2) where many meaningful different observations had been checked in callus and regeneration and the agar levels studied, that is to say the agar level treatments in 0,8% presented a major number of regeneration
Macroalgas marinhas constituem um recurso vital para a economia de diversos países e tem sido exploradas ao redor do globo devido a sua capacidade de produção de fico colóides como o ágar, carragenina e alginatos, bem como pelo seu espectro de utilização, seja na indústria de alimentos, de fertilizantes e na medicina. Estudos sobre a indução, cultivo e reorganização de calos, tem representado um importante papel nas técnicas de cultura de tecidos e suas aplicações. Em diferentes tecidos cultivados in vitro, a utilização de reguladores de crescimento é de importância primordial para o estabelecimento da competência e determinação, condições necessárias para a formação de calos e regenerações. No presente estudo foram testados os efeitos de uma auxina e de uma citocinina, em diferentes concentrações de ágar em meio ASP 12-NTA. O efeito do ácido indolacético (AIA) e da 6-benzilaminopurina (BAP) foram testados em explantes de macroalgas de espécie Gracilaria birdiae, em separado, com duas concentrações de 0,5 mg/L e 0,8 mg/L e em conjunto, nas concentrações de 1:5 mg/L e 5:1 mg/L. Com relação a concentração de ágar, foram testadas duas concentrações, com 0,5% e 0,8% para todos os tratamentos. Para o processo de esterilização foram aplicados um fungicida (nistatina) e um antibiótico (ciprofloxacina), além de iodopovidona e hipoclorito de sódio. Todo o experimento foi conduzido em câmara de germinação, com temperatura de 25 ± 2oC e fotoperíodo de 16hs de luz e 08hs escuro, salinidade de 30 ± 2‰ e pH em torno de 7,6, durante aproximadamente 60 dias. Ao final de 50 dias de cultivo, foi observada a formação de calos e de processos de regeneração indireta dos mesmos. Os resultados foram submetidos a tratamentos estatísticos de análise de contigência através do Quiquadrado (χ2 ), onde foram observadas diferenças significativas entre a incidência de calos e regenerações e os níveis de ágar estudados. Ou seja, nos tratamentos onde foram utilizados níveis de ágar de 0,8%, apresentaram o maior número de regenerações

Orientador: Lucia Helena Sipaúba Tavares
Banca: Maria Stela Maioli Castilho Nol
Banca: Claudinei da Cruz
Resumo: A microalga A. gracilis é conhecida pela sua capacidade de ser usada em nutrição de organismos aquáticos. Alguns estudos relatam que a adição de vitamina junto ao meio de cultura promove uma melhora nas condições do cultivo. O objetivo deste estudo foi avaliar o desempenho da microalga Chlorophyceae, Ankistrodesmus gracilis, em condições controladas em três diferentes meios de cultura, sendo um comercial (CHU12) e dois alternativos (NPK (20:5:20) e Macrófita + NPK) durante o período de 28 dias de cultivo para cada meio. O experimento foi realizado em recipientes de 13L durante 28 dias, com luminosidade constante de 60 μmol m-2 s-1 e para cada recipiente foram adicionados 20 mg de vitamina do complexo B. No meio CHU12 apresentou densidade celular máxima de 25,6 x 105 cel mL-1 e nos meios NPK e M+NPK foi de 25,5 e 24,2 x 105 cel mL-1 , respectivamente. As quantidades de lipídios obtidas no presente estudo foram, para o meio NPK o valor médio foi de 12,68% do peso seco e para os meios M + NPK e CHU12 os valores obtidos foram 9,85 e 2,82% do peso seco respectivamente. Em relação às proteínas a microalga A. gracilis apresentou valores significativos comprovando assim o seu grande potencial como nutriente na alimentação de organismos aquáticos, foram obtidos para o meio NPK valores de 41,26% do peso seco, e para os meios M + NPK e CHU12 valores de 37,71 e 31,76% do peso seco respectivamente. Através dos dados obtidos neste estudo, pode-se concluir que a utilização dos meios de cultura alternativos NPK (20:5:20) e macrófita + NPK apresentaram resultados satisfatórios no crescimento e desenvolvimento da microalga A. gracilis. Em relação à nutrição de organismos aquáticos pode ser uma grande ferramenta como alimento natural
Abstract: The microalgae A. gracilis is known for its ability to be used in nutrition of aquatic organisms. Some studies report that the addition of vitamin on the culture medium promotes an improvement in the conditions of cultivation. The objective of this study was to evaluate the performance of microalgae Chlorophyceae, Ankistrodesmus gracilis, under controlled conditions in three different culture media, being a commercial (CHU12) and two alternatives (NPK (20:5:20) and Macrophyte+NPK) during the period of 28 days of cultivation for each half. The experiment was accomplished in 13L containers during 28 days, with constant brightness of 60 μmol m-2 s-1 and for each container were added 20 mg of vitamin B complex. In the middle CHU12 has maximum cell density 25,6 x 105 cel mL-1 and NPK and M+NPK media was 25,5 and 24,2 x 105 cel mL-1, respectively. The quantities of lipid obtained in this study were, in the middle the average value was NPK 12,68% of the dry weight and the means M + NPK and the values obtained CHU12 were 9,85 and 2,82% of the dry weight respectively. In relation to protein microalgae A. gracilis presented significant values proving so its great potential as a nutrient in the feeding of aquatic organisms, were obtained for the NPK values of 41,26% of dry weight, and for the means CHU12 37,71 values and M+NPK and 31,76% of the dry weight respectively. Through the data obtained in this study, it can be concluded that the use of alternative culture media NPK (20:5:20) and macrophyte + NPK presented satisfactory results in the growth and development of microalgae A. gracilis. In relation to the nutrition of aquatic organisms can be a great tool as health food
Mestre

Detailed analyses of the lipid class and fatty acid composition were carried out for the marine microalgal species Nannoch/oropsis oculata (Droop) (CCAP strain no. 849/1) of the division Eustigmatophyceae (Hibberd). The alga was grown in batch and continuous culture using a novel culturing apparatus, the cage culture turbidostat, the construction of which is detailed in full. The total lipid extract yielded by the alga varied in a growth-phase dependent manner within the range 25 % to 80 % of the lyophilised cell mass. Of this between 40 % and 70 % was recovered as fatty acid methyl esters (FAME) upon transesterification. The total fatty acid composition of N. oculata consisted mainly of 16:0, 16:1 and 20:5(n-3), these three fatty acids often accounting for greater than 80 % of the total fatty acid mass. Between 9 % and 50 % of the mass of total FAME was accounted for by 20:5(n-3), the balance being accounted for by variations in the relative proportions of 16:0, 16:1, 18:1, 18:2 and 20:4. During periods of low cellular division rate, such as the lag- and stationary-phases, the proportion of polyunsaturated fatty acids (PUFA) (mainly 20:5(n-3» decreased. The total fatty acids became increasingly saturated as higher proportions of shorter chain length fatty acids accumulated, mainly in triacylglycerols (TAO). Increased cellular proportions of total lipid resulted from TAO accumulation which occurred on account of preferential partitioning of carbon into TAO biosynthesis whilst cellular division was suspended. The fatty acid composition of the TAO was more saturated at high synthesis rate and vice-versa at lower rates. The galactolipids, monogalactosyldiacylglycerol (MODO) and digalactosyldiacyl glycerol (DODO) were rich in 20:5(n-3) during exponential cell division containing up to 77 % and 53 % 20:5(n-3) respectively. Phosphatidylcholine (PC) was the only cl~s to contain significant proportions of CIS fatty acids during exponential growth, thus implicating its involvement in the acyl chain elongation reactions between the Cl6 and C20 fatty acids. Culture incubation temperature in the range 5 °C to 25°C did not influence the fatty acid composition of N. oculata. The effect of temperature upon culture dynamics at the lower culture incubation temperatures gave an apparent decrease in the PUFA content of the total fatty acid at a given point on the cultures growth curves. By expressing the data in tenns of culture doubling periods during the exponential-phases of growth it was found that temperature had no real effect upon fatty acid unsaturation or chain length. at either the total or the individual lipid class FAME level after the cells had passed through five doubling periods. Increasing the culture medium salinity from one quarter to one and a half times that of normal seawater decreased the un saturation and chain length of the fatty acids at both total and individual lipid class levels. The change resulted from the progressive accumulation of 18:1 and 18:2"at the expense of 20:5. Variation of salinity did not affect the dynamics of the cultures in the same respect as temperature in that a lag-phase was not observed on the cultures growth curves. However. such a phase was evident in the fatty acid profile of the cells in the period following inoculation. The 'effects of culture illumination intensity in the range 45 Jill m-2 sec-I to 170 Jill m 2 sec-! were examined under continuous culture conditions using the cage culture turbidostat Accumulation of saturated TAG by the cells at the higher illumination intensities gave an apparent decrease in the rate of PUFA biosynthesis. The polar lipid classes were found to be more highly unsaturated at higher illumination intensities. At lower illumination intensity TAG accumulation was reduced and the total fatty acid composition was accordingly more unsaturated. The fatty acid composition of the TAG component was more unsaturated but those of the polar lipid classes were less unsaturated than at higher illumination intensity. Increased illumination increased the degree of un saturation of the polar lipid cl~sses. Excess fixed carbon was partitioned into TAG biosynthesis. primarily as 16:0 and 16:1. The net accumulation of this lipid class even at high cell division rates resulted in a low overall unsaturation level. The effects of decreasing nitrate concentration in the range 1.0 mM N03 - to 0.001 mM N03 - had a similar basis to those of illumination in that the changes in the total fatty acid composition were largely governed by the rate of TAO accumulation. At high nitrate concentrations the cellular division rate was relatively high and the proportion of TAO in the total lipid extract was low. Consequently, both total and individual lipid classes contained high proportions of unsaturates, particularly 20:5(n-3). However, when the nitrate concentration was decreased, such that it began to limit the rate of cellular division, TAG accumulated Cursory analyses of the molecular species of the galactolipid classes, MODO and DODO, and phospholipid class PC are presented. The effects of environmental variables are discussed in tenns of the changes which may occur in the growth phase distribution of the cells in asynchronous culture, along with the concommitant changes in the lipid composition of the cells. The potential linkage of the elongation and desaturation reactions with both MODO and PC is also discussed briefly with reference to future research.