O cultivo de microalgas é uma matéria prima para produção de biocombustível e de captura de carbono devido a vantagens como alta produção de biomassa e rápido crescimento quando comparado com outras fontes de energia e não necessitar de terra fértil. O presente trabalho teve como objetivo estudar métodos de concentração da biomassa. A microalga utilizada foi a Isochrysis galbana. Os cultivos tiveram duração de 20 dias e concentração inicial de 7.104 cel/mL no meio de cultivo F2/Guillard. e foram realizados em fotobioreatores de 500 mL, 3 L e 12 L. Os experimentos foram conduzidos em foto-período de 12 h claro/escuro, com temperatura de 27 a 29 C. Ao final dos cultivos, as amostras foram levadas para a sequência de processos de separação. Inicialmente, foram realizados ensaios de microfiltração em membrana com porosidade de 0,45 m em procedimento do tipo dead-end e constatou-se a rápida e intensa formação de camada de fouling. Acrescentou-se uma etapa de separação por floculação preliminar à microfiltração, utilizando-se Al2(SO4)3 como agente floculante. O meio coagulado foi então filtrado e microfiltrado. O estudo combinado das 3 etapas de separação possibilitou 99% de remoção de biomassa.O teor de óleo obtido foi de 22,4%. Portanto, o trabalho apresenta uma configuração de concentração da biomassa Isochrysis galbana visando o processo de produção de biocombustíveis / The cultivation of microalgae is a feedstock for biofuel production and carbon sequestration due to advantages such as high-biomass production and, fast growth when compared to other energy sources and does not require fertile land. This work aimed to study methods for biomass concentration. The microalgae used was Isochrysis galbana. The cultivation lasted 20 days and initial concentration of 70,000 cells / mL F2/Guillard culture medium and were performed in. Photobioreactors of 500 mL, 3 L and 12 L. The experiments were conducted in photoperiod of 12 h light / dark, temperature of 27-29 C. At the end of cultivation, samples were taken for the sequence of separation processes Initially, tests were performed on microfiltration membrane with 0.45 μm pore diameter in a dead-end procedure, and the rapid and intense formation of fouling layer was noted. One step of preliminary separation by flocculation previous to microfiltration was added to the process, using Al2(SO4)3 as flocculant agent. The coagulated medium was then filtered and microfiltered. The combined study of three separation steps allowed 99% removal of biomassa.O oil content obtained was 22.4%. Therefore, the work presents a configuration in biomass concentration of Isochrysis galbana aiming at the process of production of biofules

Microalgas e cianobactérias têm sido amplamente recomendadas para biomonitoração de metais pesados e outros poluentes, sendo considerados indicadores sensíveis às alterações ambientais e utilizados como organismos testes na regulamentação dos níveis de metal. Estes micro-organismos fotossintetizantes são produtores primários da base da cadeia alimentar aquática e são os primeiros a serem afetados pela poluição por metais pesados. O cobre é um metal normalmente considerado como nutriente essencial para a vida aquática mas pode ser tóxico para algumas espécies. Portanto, neste estudo foram avaliados o efeito tóxico e a bioacumulação de cobre (II) em quatro espécies de micro-organismos fotoautotróficos componentes do fitoplâncton dulcícola, duas cianobactérias filamentosas (Anabaena sp. e Oscillatoria sp) e duas microalgas da classe das clorofíceas (Monorraphidium sp. e Scenedesmus sp.). O meio de cultivo utilizado nos ensaios foi o ASM-1 com e sem a presença de cobre (0,6 mg/L a 12 mg Cu2+/L) onde, o efeito tóxico do metal foi monitorado por contagem celular para as microalgas e por peso seco para as cianobactérias. A bioacumulação do metal foi avaliada da mesma forma para todos os micro-organismos, através de coletas de amostras no decorrer do experimento e determinação da concentração de cobre em solução por espectrometria de absorção atômica com chama. Os resultados obtidos mostram que o efeito tóxico do metal é diretamente proporcional à concentração inicial para os micro-organismos estudados, mas que o cobre (II) foi mais tóxico para as cianobactérias que para as microalgas verdes. A bioacumulação teve uma relação direta com o efeito tóxico do metal sobre os micro-organismos. Os resultados obtidos permitem sugerir que cobre (II) tem efeito negativo no fitoplâncton, inibindo o crescimento e alterando parâmetros metabólicos como a fotossíntese. A bioacumulação do metal pode comprometer os níveis tróficos da cadeia alimentar, afetando seu transporte para seres superiores / Microalgal and cyanobacterial cells are widely used in the biomonitoring of metal contaminated areas as well as other pollutant agents, being considered sensitive microorganisms in relation to environmental changes and also used as test-organisms in the regulation of low levels of metals. These photosynthesizing microorganisms are primary producers in the aquatic food chain, and are the first ones to be affected by heavy metal pollution. Copper is a metal usually considered as an essential nutrient for aquatic forms of life, however toxic for some species. In this way, in the present study, the toxic effect of copper ion and its bioaccumulation by photoautotrophic microbes from freshwater phytoplankton: two filamentous cyanobacteria (Anabaena sp. and Oscillatoria sp) and two chlorophyte microalgae Monorraphidium sp. and Scenedesmus sp.). The culture medium used in the tests was the ASM-a medium in absence and contaminated with copper ion (0,6 mg/L to 12 mg Cu2+/L). The toxic effect of copper was monitored through cell count (microalgal cells) and dry weight measurements (cyanobacterial cells), and copper bioaccumulation was evaluated for all the species tested, through determination of the residual metal concentration in solution, by flame atomic absorption spectrometry. Results obtained indicated that the toxic effect of the metal was directly proportional to the initial concentration of the ion for all the species tested; however, copper ion was more toxic to cyanobacterial than to microalgal cells. The bioaccumulation of copper was directly related to the toxic effect of the ion over the microorganisms. Results obtained suggest that copper ion has a negative effect on the phytoplankton, inhibiting cell growth and also affecting metabolic parameters such as photosynthesis. The bioaccumulation of the metal can markedly affect the trophic levels in the food chain, thus affecting its transport to higher species

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-06T13:45:38Z No. of bitstreams: 2 Dissertação - Eliéser Viégas Wendt - 2014.pdf: 2872867 bytes, checksum: 608311a9abf7ecb2f1a1fce9a77164d5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-19T13:50:39Z (GMT) No. of bitstreams: 2 Dissertação - Eliéser Viégas Wendt - 2014.pdf: 2872867 bytes, checksum: 608311a9abf7ecb2f1a1fce9a77164d5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-19T13:50:46Z (GMT). No. of bitstreams: 2 Dissertação - Eliéser Viégas Wendt - 2014.pdf: 2872867 bytes, checksum: 608311a9abf7ecb2f1a1fce9a77164d5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The purpose of this study was to determine the composition of chemical elements in freshwater microalgae evaluated as feedstock for biodiesel production in order to assess possible consequences on the biodiesel quality and to determine whether the use of residual biomass as animal feed. So the 26 chemicals elements in 76 freshwater algae samples, 39 microalgae biomass obtained once direct transesterification, 6 oilseeds, one sample of beef tallow as well as biomass, oil and biodiesel of microalgae Desmodesmus ssp. were measured by ICP-OES. Microalgae stood out compared to conventional feedstock due to higher contents of Na and Fe, moreover chemical elements such as Al, B, Cu, Mn, Sn, Zn and as Fe, are not monitored and may be existing in microalgae biodiesel. Thru direct transesterification procedure, most of the macronutrients constituents of microalgae is lost. Residual biomass by solvent extraction process keeps the mineral composition and in this case, is the most suitable for animal feed. The contents of chemical elements from Desmodesmus ssp. biomass decrease throughout the biodiesel production process by direct transesterification. However, the P content in biofuel was 127 mg/kg, about 13 times beyond the specification, so it being necessary the pretreatment consequence to proceed the transesterification. The study demonstrated that standardization of biodiesel quality control should include the control of other chemicals, further those already monitored, if biodiesel is mainly derived from microalgae feedstock. / O objetivo deste trabalho foi avaliar a composição de elementos químicos em microalgas dulcícolas avaliadas como matéria-prima para produção de biodiesel visando estimar possíveis consequências na qualidade do biodiesel e determinar a possibilidade de aproveitamento da biomassa residual como ração animal. Para isso foram determinados por ICP-OES os teores de 26 elementos químicos em 76 amostras de microalgas dulcícolas, 39 biomassas das microalgas obtidas após procedimento de transesterificação direta, 6 sementes de oleaginosas, 1 amostra de sebo bovino e, finalmente, biomassa, óleo e biodiesel obtido a partir da microalga Desmodesmus ssp. As microalgas destacaram-se em relação às matérias-primas convencionais pelos elevados teores de Na e Fe. Também foram encontrados elementos químicos como Al, B, Cu, Mn, Sn, Zn e que, assim como o Fe, não são monitorados e poderão estar presentes no biodiesel de microalgas. Durante o procedimento de transesterificação direta, boa parte dos macronutrientes constituintes da microalga é perdida. Já a biomassa residual do processo de extração por solvente mantém a composição mineral e, neste caso, é a mais indicada para a utilização na alimentação animal. Os teores dos elementos químicos da biomassa da microalga Desmodesmus ssp. diminuem ao longo do processo de obtenção de biodiesel via transesterificação direta. Entretanto, a concentração de P no biocombustível foi de 127 mg/kg, cerca de 13 vezes acima do especificação, sendo necessário neste caso, efetuar o tratamento do óleo via degomagem antes de proceder a transesterificação. O trabalho demonstrou que a normalização do controle de qualidade de biodiesel deve contemplar o controle de outros elementos químicos, além dos já monitorados no biodiesel, principalmente se o biodiesel for obtido a partir de microalgas como matéria-prima graxa.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Embora todas as etapas do processo de produÃÃo de biodiesel a partir de microalgas sejam essenciais, a separaÃÃo e a ruptura celular da biomassa sÃo particularmente importantes, uma vez que as tecnologias disponÃveis para este fim apresentam elevados custos, comprometendo a viabilidade do aproveitamento energÃtico. Este trabalho teve como objetivo apontar um sistema que conseguisse aliar a separaÃÃo da biomassa algal de efluente de sistemas de lagoas de estabilizaÃÃo à ruptura celular, como uma alternativa de prÃ-tratamento para maximizar a extraÃÃo de lipÃdios pelo mÃtodo modificado de Bligh e Dyer; assim como estudar preliminarmente a comunidade fitoplanctÃnica presente para comparaÃÃo dos rendimentos lipÃdicos. Um reator de eletroflotaÃÃo nÃo-convencional foi confeccionado para operar em batelada e em fluxo contÃnuo, utilizando-se eletrodos nÃo consumÃveis e baixa potÃncia elÃtrica. A metodologia proposta foi comparada com tÃcnicas tradicionalmente utilizadas para ruptura celular: autoclave, micro-ondas e ultrassom. Foram selecionadas seis estaÃÃes de tratamento de esgotos (ETE) com diferentes configuraÃÃes de lagoas de estabilizaÃÃo para anÃlise dos gÃneros fitoplanctÃnicos dominantes e do teor lipÃdico. Observou-se a separaÃÃo da biomassa, sendo obtida eficiÃncia de remoÃÃo de turbidez superior a 80% em 20 minutos de operaÃÃo do reator em batelada. Entre os mÃtodos de prÃ-tratamento tradicionais testados, o micro-ondas foi o que apresentou maior rendimento lipÃdico (33,7  5,3%), diferente estatisticamente da autoclave (15,4  2,26%) e ultrassom (13,3  2,96%). Para a eletroflotaÃÃo nÃo-convencional o rendimento lipÃdico foi de 24,8  7,05%, que nÃo apresentou diferenÃa estatÃstica quando comparado ao micro-ondas. Entretanto, quando os custos energÃticos foram considerados, a eletroflotaÃÃo nÃoconvencional apresentou um custo de 5,6 Wh.g-1, sendo menor que o obtido para o micro-ondas. Os gÃneros de microalgas que predominaram nos efluentes analisados pertenciam Ãs classes Chlorophyceae, Cyanophyceae e Euglenophyceae. Em uma anÃlise quantitativa geral, os efluentes de todas as estaÃÃes analisadas apresentaram rendimento lipÃdico entre 8,5 e 34,6%. Os efluentes das ETE Aquiraz e TupÃ-Mirim apresentaram os maiores teores lipÃdicos (28,4  6,3% e 23,1  3,6%, respectivamente), sem diferenÃa estatÃstica (p=0,12). Os efluentes das ETE Araturi e Tabapuà apresentaram os menores potenciais lipÃdicos, sem diferenÃa estatÃstica entre eles (14,3  5,9% e 15,6  4,9%, respectivamente, p=0,68). A eletroflotaÃÃo nÃo-convencional mostrou-se como uma metodologia promissora para separaÃÃo e ruptura das cÃlulas de microalgas de efluentes de lagoas de estabilizaÃÃo, cujos potenciais lipÃdicos assemelharam-se aos obtidos a partir de biomassa microalgal cultivada em processos convencionais. AlÃm disso, a metodologia desenvolvida neste trabalho soma um motivo para a separaÃÃo e utilizaÃÃo da biomassa algal de lagoas de estabilizaÃÃo evitando, com isso, seu lanÃamento nos corpos dÂÃgua e garantindo um ganho econÃmico e ambiental, uma vez que visa o aproveitamento da biomassa para a produÃÃo de biocombustÃvel, ao passo que minimiza impactos ambientais decorrentes da presenÃa elevada desse material no ambiente. / Although all stages on the production of biodiesel from microalgae cells are essential, harvest and cell disruption biomass are particularly important, since the available technologies for this purpose present high cost, compromising energy recovery viability. This study aimed to develop a system that could combine separation of algal biomass from wastewater stabilization pond systems to cell disruption as an alternative of pretreatment to maximize total lipids extraction by the modified method of Bligh and Dyer, as well as perform a preliminary study of the phytoplankton for comparison of lipid content. A non-conventional electroflotation reactor was designed to operate in batch and continuous flow, using non-consumable electrodes and low electrical power. The proposed methodology was compared to traditional techniques used for cell disruption: autoclave, microwave and ultrasound. Six Wastewater Treatment Plant (WTP) with different pond configuration were selected for analysis of the dominant phytoplankton genera and lipid content. Biomass separation was achieved, with turbidity removal efficiency exceeding 70% in 20 minutes of operation with the batch reactor. Among the traditional pretreatment methods tested, the microwave showed the highest lipid yield (33.7  5.3%), followed by autoclave (15.4  2.26%) and ultrasound (13.3  2.96%). For non-conventional electroflotation methodology, lipid yield was 24.8  7.05%, which showed no statistical difference when compared to the microwave method. However, when energy cost was considered, non-conventional electroflotation presented a cost of 5.6 Wh.g-1, which was smaller than that obtained by the microwave method. The predominant microalgae genera in the analyzed effluents belonged to the classes Chlorophyceae, Cyanophyceae and Euglenophyceae. In a general quantitative analysis, the effluents from the analyzed ponds presented lipid yield between 8.5 and 34.6%. Effluents of the WTP Aquiraz and TupÃ-Mirim achieved the highest lipid content (28.4  6.3% and 23.1  3.6%, respectively), with no statistical difference (p = 0.12). Effluents of the WTP Araturi and Tabapuà achieved the lowest lipidic potential, with no statistical difference (14.3  5.9% and 15.6  4.9%, respectively, p = 0.68). Non-conventional electroflotation proved to be a promising methodology for harvesting and cell disruption of microalgae provided by effluent stabilization ponds, whose potential lipid resembled those obtained from microalgal biomass cultivated in conventional processes. Furthermore, the methodology developed in this work adds a reason for the harvesting and utilization of algal biomass from waste stabilization ponds avoiding, thus, its release into water bodies and ensuring an environmental and economic gain, since it aims the use of biomass for biofuel production, while minimizing environmental impacts due to elevated presence of this material on the environment.

Diversos produtos tópicos são desenvolvidos a fim de se evitar os efeitos nocivos da radiação ultravioleta (UV), dentre eles, os protetores solares constituídos por filtros físicos e/ou químicos. No entanto, os filtros químicos podem promover eventos adversos, razão pela qual a busca de ativos em fontes naturais tem-se mostrado de grande importância. A microalga Botryococcus braunii pode ser de potencial interesse em aplicações cosméticas, pois foi descrito que seu extrato aquoso apresentou redução da desidratação cutânea, induziu a produção do colágeno, bem como promoveu atividade antioxidante. Assim, o estudo visou analisar o auxilio da B. braunii na fotoproteção cutânea e na proteção da pele por meio da ação antioxidante in vitro. As formulações, constituídas de biomassa da microalga ou de seus extratos, acrescidas ou não de filtros, foram avaliadas quanto à: ação antioxidante, atividade fotoprotetora in vitro e fotoestabilidade. A B. braunii apresentou bom desempenho de crescimento, contendo lipídeos (25,65%) e proteínas (49,17%) em proporção semelhante à descrita na literatura. A presença de fenóis totais e a ação antioxidante foram analisadas nos extratos obtidos com clorofórmio, metanol e etanol 70%. O extrato obtido com etanol 70% foi o que apresentou maior concentração de fenóis totais (68,9 µg/mL de ácido cafeico), porém inferior ao relatado na literatura; e a ação antioxidante não foi considerada significativa ao se comparar com o padrão Trolox®. Nas formulações, os extratos foram considerados ineficientes para ação antioxidante. Quanto à atividade fotoprotetora in vitro e fotoestabilidade, a B. braunii não apresentou eficácia adequada, existindo, porém, a possibilidade de os extratos atuarem na região da luz visível e contribuírem contra os efeitos danosos provocados por esta radiação à pele. / Several topical products are developed in order to avoid the harmful effects of ultraviolet (UV) radiation, including sunscreens made by physical and/or chemical filters. However, chemical filters may promote adverse events, which is why the search for actives from natural sources has proven to be of great importance. The microalgae Botryococcus braunii may be of potential interest in cosmetic applications, because it has been reported that its aqueous extract showed a reduction in skin dehydration, induced collagen production and promoted antioxidant activity. Thus, the study aimed to analyze the contribution of B. braunii in skin photoprotection and skin protection through in vitro antioxidant action. The formulations, consisting of biomass from the microalgae or their extracts, supplemented or not by filters, were evaluated for: antioxidant activity, photoprotective activity in vitro and photostability. The B. braunii showed good growth performance, containing lipids (25.65%) and protein (49.17%) in similar proportion to the described by the literature. The presence of phenolic compounds and antioxidant activity were analyzed in extracts obtained with chloroform, methanol and 70% ethanol. The extract obtained with 70% ethanol showed the highest concentration of total phenolics (68.9 mg/mL caffeic acid), but lower than that reported in the literature; and the antioxidant action was not considered significant when compared to the Trolox® standard. In the formulations, the extracts were considered inefficient for antioxidant action. In relation to the in vitro photoprotection activity and photostability, B. braunii did not provide adequate efficacy, however, there is a possibility that the extracts have an ability to act in the visible light region and contribute to the protection from the harmful effects caused by that radiation to the skin.

A necessidade de se encontrar um substituto ao combustível fóssil tem impulsionado o desenvolvimento de novas fontes de biomassa para os biocombustíveis. Atualmente uma fonte de biomassa alternativa que vêm ganhando destaque são as microalgas, micro-organismos fotossintéticos capazes de capturar o CO2 atmosférico e acumular altos teores de lipídeos em sua biomassa, tornando-os muito atrativos como fonte de matéria-prima para a síntese de biodiesel. Deste modo, o presente trabalho teve como objetivo estudar os efeitos dos fatores NaNO3, CO2, intensidade luminosa e profundidade no cultivo da microalga marinha Chlorella sp visando o acúmulo de lipídeos dessa microalga em sua biomassa para a produção de biodiesel. O trabalho foi dividido em três etapas principais: na primeira foram realizados os cultivos nos quais os fatores de interesse foram avaliados em dois níveis de operação, NaNO3 (0,25 e 0,75 g/l), CO2 (5 e 10 %), intensidade luminosa (0,85 e 14,5 klux) e profundidade (5 e 10 cm); na segunda etapa foram realizadas as extrações lipídicas da biomassa para a quantificação dos teores lipídicos; e na terceira etapa realizou-se a síntese do biodiesel por via química a partir dos lipídeos extraídos da biomassa. Os experimentos que compreenderam a etapa dos cultivos foram realizados segundo um planejamento de Taguchi L8, considerando como variáveis respostas concentração de biomassa e teor lipídico. O melhor ajuste para maximizar a concentração de biomassa e teor lipídico foi de 0,85klux de intensidade luminosa, 5% de CO2, 10 cm de profundidade e 0,42g.L-1 de NaNO3 que forneceu uma concentração de biomassa de 2,04 g.L-1 com 15,04% de teor lipídico. O perfil lipídico apresentou uma proporção de 60 % entre ácidos graxos saturados, 31,7% entre ácidos monoinsaturados e 8,26 % de ácidos poli-insaturados e a reação de síntese do biodiesel atingiu uma conversão de 78,4%. / The necessity of finding a substitute to fossil fuel has enhanced the development of new biomass sources for biofuels. Currently, an alternative source of biomass that has acquiring prominence are microalgae, which are photosyntetical microorganisms able to capture the atmospheric CO2 and accumulate high content of lipids in their biomass, making them a very attractive feedstock source for biodiesel synthesis. In such a way, the present work had the objective of studying the effect of the following factors: NaNO3, CO2, luminous intensity and depth in the cultivation of marine microalga Chlorella sp, aiming the lipid accumulation of this microalga in its biomass for biodiesel production. The project was divided in three main steps: in the first one, the experiments were performed to evaluate the following factors in two levels of operation, NaNO3 (0,25 and 0,75 g/l), CO2 (5 and 10 %), luminous intensity (0,85 and 14,5 klux) and depth (5 and 10 cm); in the second step, the lipid extractions from the biomass were performed to quantify the lipid contents; and in the third one, the synthesis of biodiesel via chemical route was performed utilizing the lipids extracted from the biomass. The experiments regarding the cultivation steps were performed according to a Taguchi L8 experimental design, considering the biomass concentration and lipid content as response variables. The best fit for maximization of biomass concentration and lipid content was 0,85 klux of luminous intensity, 5% of CO2, 10 cm of depth and 0,42g/l of NaNO3, which provided a biomass concentration of 2,04 g/l with 15,04% of lipid content. The lipid profile showed a proportion of 60% of saturated fatty acids, 31,7% of monounsaturated fatty acids and 8,26% of polyunsaturated fatty acids and the reaction of biodiesel synthesis yielded a conversed of 78,4%.

O objetivo desta tese foi explorar o sistema de produção de proteínas heterólogas em microalga com ênfase em Chlamydomonas reinhardtii por meio de: (1) desenvolvimento de um fotobiorreator tubular fechado de escala laboratorial, utilizando técnicas de manufatura digital; (2) avaliação de 7 diferentes proteínas fluorescentes (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato e mCherry), como sistema reporter de secreção de proteínas em microalga; (3) avaliação do fotobiorreator desenvolvido utilizando cultivo de cepas recombinantes; (4) desenvolvimento de novos peptídeos sinais para secreção de proteínas em C. reinhardtii; (5) avaliação da produção de um biofármaco (hialuronidase) em microalgas, por meio da expressão de duas isoenzimas codificadas pelos genes HYA1 e SPAM1 em C. reinhardtii. O fotobiorreator tubular foi avaliado quanto a sua capacidade de resistir ao processo de esterilização por autoclavação e seu desempenho por meio do cultivo de cepa recombinante secretando mCherry. A fluorescência das proteínas fluorescentes foi medida por leitor de placas de fluorescência e visualizada intracelularmente por microscopia confocal de fluorescência. A atividade de hialuronidase foi determinada através de um ensaio enzimático turbidimétrico. O desenvolvimento do fotobiorreator resultou em um sistema fechado resistente a autoclavação, com capacidade de cultivo de cepas recombinantes de C. reinhardtii. Esse fotobiorreator proporcionou uma produtividade máxima de 10 mg/L.d de mCherry da cepa recombinante em sistema fechado, com velocidade específica de crescimento máxima de 1,27 d-1 para a cepa recombinante testada. Todas as proteínas fluorescentes avaliadas apresentaram capacidade de secreção por C. reinhardtii, com diferentes níveis de interferências em sua medição, permitindo a escolha da mCherry como proteína reporter. Entre os peptídeos sinais avaliados (quatro descritos na literatura - BiP, ARS1, CAH1 e IBP1 - e seis preditos), o peptídeo predito \"SP5\" foi o que apresentou maior capacidade de secreção, determinado por níveis de fluorescência no sobrenadante. A avaliação dos peptídeos sinais constatou a necessidade de explorar o desenvolvimento de sistemas de expressão (e.g. vetores de expressão) aliados a análises computacionais, como o SignalP 4.0. Por último, os dados desse estudo mostram que C. reinhardtii transformadas com o vetor de expressão foi capaz de produzir as duas isoformas de hialuronidase em sua forma ativa, evidenciando a capacidade desse sistema para a produção de biofármacos. Portanto, nesta tese o sistema de expressão de proteínas heterólogas baseado em microalgas foi explorado, atingindo os objetivos propostos. O fotobiorreator desenvolvido tem a capacidade de esterilização em escala laboratorial (1) e em cultivo com cepa recombinante propiciou elevada produtividade (3). As proteínas vermelhas fluorescentes apresentaram-se como as proteínas com menores interferências para estudos de secreção em C. reinhardtii (2). Além disso, o peptídeo predito SP5 apresentou o melhor desempenho na secreção de proteínas (4) e o vetor de expressão empregado permitiu a identificação de cepas produtoras de biofármaco hialuronidase (5). Portanto, o sistema de produção de proteínas heterólogas por microalgas é um sistema promissor e poderá permitir, utilizando sistemas de secreção, obter proteínas de alto valor comercial a baixos custos, empregando a secreção e técnicas de cultivo como a fermentação extrativa. / In this thesis, the heterologous protein production in microalgae with emphasis on Chlamydomonas reinhardtii was explored through: (1) development of a laboratory scale closed tubular photobioreactor using digital manufacturing techniques; (2) evaluation of different fluorescent proteins (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato and mCherry) as a reporter system for protein secretion in microalgae (3) evaluation of photobioreactor developed using recombinant strains culture; (4) development of new signals peptides for protein secretion in C. reinhardtii (5) expression evaluation of a biopharmaceutical (Hyaluronidase) in microalgae, through the expression of two isoenzymes encoded by the HYA1 and SPAM1 genes in C. reinhardtii. The tubular photobioreactor was evaluated for its ability to resist sterilization process by autoclaving and its performance by culturing recombinant strain secreting mCherry. Fluorescence of fluorescent proteins was measured by fluorescence plate reader and observed intracellularly by confocal fluorescence microscopy. The hyaluronidase activity was determined by a turbidimetric enzymatic assay. The development of the photobioreactor resulted in a closed system resistant to autoclaving, capable of culturing recombinant strains of C. reinhardtii. This recombinant strain achieved a maximum productivity of 10 mg/L.day of mcherry in the closed system, with a maximum growth rate of 1.27 d-1 for the recombinant strain tested. All the fluorescent proteins evaluated had C. reinhardtii secretion capacity, with different interference levels in their measurement, allowing the selection of mCherry as a reporter protein. Among the evaluated peptides (four described in the literature - BiP, ARS1, CAH1 and IBP1 - and six predicted), the predicted peptide \"SP5\" was the one that presented greater capacity of secretion, determined by levels of fluorescence in the supernatant. The results of this study point out the need to explore the development of biological systems (i.e., expression vectors) allied to computational analysis. Finally, the data from this study showed that C. reinhardtii could produce the two isoforms of hyaluronidase in its active form, evidencing the capacity of this system to produce biopharmaceuticals. Therefore, in this thesis the heterologous protein expression system based on microalgae was explored, reaching the proposed objectives. The developed photobioreator has sterilization capabilityin laboratorial scale (1) and in culture with recombinant strain had high productivity (3). The red fluorescent proteins was found as the most suitable proteins for studies of secretion in C. reinhardtii with lower interference levels(2). In addition, the predicted SP5 peptide showed the best performance in protein secretion (4) and the expression vector employed allowed the identification of strains producing biopharmaceutical hyaluronidase (5). Therefore, the system of heterologous proteins production by microalgae is promising and will allow, using secretion systems, to obtain proteins of high commercial value at low costs, using secretion and cultivation techniques such as extractive fermentation.

Global warming is becoming more concerned by the public. The escalating atmospheric CO₂ level has introduced the intensification of greenhouse effect which brought enormous impact to the environment and climate. Among different methods for CO₂ mitigation, biological treatment on CO₂ emission using microalgae is regarded to be more economical and beneficial. On the other hand, countries around the world are projected to face water scarcity in the coming decades. Therefore alternatives to the finite fresh water resources for consumption have to be explored. Seawater has been introduced for toilet flushing in Hong Kong since 1950s. The flushing water is mixed with the consumed fresh water in the sewage treatment process to give a sewage effluent with high salinity, which still contains nutrients to support the growth of algal cells. Using sewage effluent as an algal culture medium not only can have a lower operation cost while saving fresh water for food crops, but also the effluent can be purified before discharging. Besides Hong Kong, there is an increase in numbers of cities incorporating seawater in toilet flushing systems as an economical and sustainable solution to fresh water scarcity. / Taking sustainable development into consideration, the use of algal cell for the fixation of CO₂ in saline sewage effluent is proposed, which should be an effective mitigation for CO₂ emissions, removal of nutrient in sewage effluent as well as production of useful products such as biofuel feedstock. In order to find out a suitable algal species for CO₂ the bioremediation, commercially available algal strains were compared against the locally isolated species on the growth and CO₂ removal efficiency in saline sewage effluent. Chlorogonium capillatum, the algal strain isolated from a local fish pond, was found to be the best candidate for CO₂ sequestration and nutrient removal in the non-sterile saline sewage effluent since it could grow much better in the presence of other microorganisms, comparing with the majority of other algal species could not grow under this situation. / The effect of CO₂ enhancement on algal photosynthetic rate and growth was studied in terms of the change in cell number, biomass and lipid production, and the fatty acid profile. The optimisation of CO₂ mitigation was achieved by deploying the response surface methodology (RSM) approach with a model describing the change in CO₂ consumption rate being developed. In the minimal run resolution V (MR5) screening test, it was found that salinity, pH, CO₂ and PO₄³⁻-P levels were influential to CO₂ removal by C. capillatum in non-sterile sewage effluent. Further optimisation of the CO₂ consumption rate was performed using the Box-Behnken design. The results of study showed that C. capillatum was able to deliver its maximum CO₂ consumption rate at 58.96 mg L⁻¹ d⁻¹ at the optimal condition, which was very close to the ordinary condition in the average sewage. In addition, the lipid content of C. capillatum could reach 24.26±2.67% with fatty acid profile conforming to typical biodiesel composition, delivering a high potential for biofuel feedstock production. Together with a high nutrient removal rate, C. capillatum could be used to produce a promising waste-recycling oriented simultaneous treatment system. / Since the CO₂ consumption rate was not dependent to light intensity, the spectral effects on the light-enhanced algal growth and carbon sequestration were investigated to find the best culture condition and how the carbon sequestration process was being influenced. Five LED light spectra were chosen for the analysis and it was found that growth parameters and cell compositions were influenced by the colour of the light very differently. The results indicated that under the irradiation of white LED light, C. capillatum had the highest CO₂ consumption rate and lipid content. Red LED light induced the highest amount of cellular protein as well as the chlorophyll a content. However, the performance of the light dependent reaction of the red LED light culture did not show apparent improvement. Regarding the CO₂ fixation enzyme, the spectral effect on RuBisCO content was marginal and there was no obvious relationship between the light induced CO₂ consumption and solely the light induced RuBisCO content change. Chemical analysis on the algal biomass indicated the C. capillatum culture would be a suitable microbial system to mitigate CO₂ emission, remove nutrients from saline sewage effluent and produce biomass suitable for biofuel production. / This study delivers a bioremediation system which is capable of simultaneous CO₂ mitigation, nutrient removal and biofuel feedstock production with a newly isolated algal species in a waste recycling manner. The findings of this study are not limited to the application locally in Hong Kong, but hopefully all these can also be useful in similar works in other places to help with the sustainable development. / 全球暖化正越來越受到公眾關注，大氣中不斷上升的二氧化碳水平已經加劇了溫室效應，並對環境和氣候帶來了巨大的影響。在不同的二氧化碳緩減方法中，利用生物方法以微藻處理二氧化碳排放被認為是更具效率和回報價值。另一方面，世界各地已經預計會在未來幾十年面臨水荒，因此是有必須要為有限的淡水資源尋找代替品。自五十年代起，香港便引入了海水作沖廁用途。沖廁用水會在污水處理過程中混合經使用過的淡水而產生高鹽度的污水，而污水當中仍含有營養物質，以支持藻類細胞生長。使用污水作為藻類培養介質不但可以降低營運成本，並有助於節省淡水用以耕種糧食作物，而且可以在污水排出前進行純化。除香港以外，一些引入海水用於沖廁系統的城市數目正在增加。 / 考慮到可持續發展，我們提出利用微藻細胞在帶鹽污水中進行二氧化碳固定。這應該是一個有效的緩解二氧化碳排放，清除污水中養份，以及產生有價值產品的綜合方案。為了找出一種合適的微藻進行生物整治，我們比較了市面上買到的品種和於本地環境分離出來的藻種於帶鹽污水中生長和去除二氧化碳的表現。經過實驗後我們確定一種從魚塘中分離出來的綠梭藻(Chlorogonium capillatum)是能夠在未經消毒的帶鹽污水中進行碳封存和去除養份的最佳選擇，因為綠梭藻能夠在其他微生物存在下仍可以良好地生長，反觀其他大多數的藻種就不能在這情況生長。 / 我們從細胞數、生物量、脂質和脂肪酸譜的變化方面研究了二氧化碳量增強對微藻光合速率和生長的影響。我們利用了反應曲面法(Response Surface Methodology)對微藻的二氧化碳緩減進行最佳化，並將二氧化碳緩減率的變化製成模型。在條件篩選實驗，我們找出鹽度、酸鹼、二氧化碳和磷質水平是會影響二氧化碳緩減率。在隨後的二氧化碳緩減率的最佳化後，綠梭藻的最大二氧化碳緩減率為58.69微克每公升每天。研究發現綠梭藻能夠於接近平常環境條件下達成最高效的二氧化碳緩減。除此之外，綠梭藻的脂質含量可達24.26±2.67百份比。加上脂肪酸分佈符合典型的生物柴油成份和具有高度養份去除率，綠梭藻可以用來創造以廢物回收作主導的多功能的生物修復系統。 / 由於二氧化碳緩減率並不依賴於光的強度，我們進行了光譜對微藻生長和碳封存的影響的研究，以找出最佳的培養條件和了解碳吸收的過程如何被影響。我們分析了五種發光二極管光譜，發現燈光顏色對微藻的生長參數和細胞組合物有明顯的影響。結果顯示，在白色燈的照射下，綠梭藻有最高的二氧化碳緩減率和脂質含量。紅色燈引發了最高的細胞蛋白質，以及葉綠素a含量。然而，紅光並沒有明顯提升光依賴反應。關於固定二氧化碳的酶，光譜對羧化/加氧酶的數量變化效果細微。另外，羧化/加氧酶跟二氧化碳緩減率之間沒有明顯關係。從微藻生物質中的化學分析，我們認為綠梭藻是一個合適的微生物系統以達成二氧化碳的排放緩減，去除帶鹽污水中的養份和生產適用於生物燃料製造的生物質。 / 這項研究提供了一個生物修復系統，它能夠以廢品回收方式同時減少二氧化碳排放、去除營養和生產製作生物燃料的原料。這項研究的結果並不只限於香港使用，我們希望這些東西也可以應在其地方類似的工程上，為可持續發展出力。 / Lee, Kwan Yin. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-134). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

A crescente necessidade de se obter fontes de energia e materiais mais sustentáveis tem aumentado o interesse em processos baseados na conversão de biomassa. Microalgas são particularmente interessantes por não competirem com alimentos e serem facilmente adaptáveis ao meio de crescimento. Além disso, microalgas podem ser processadas termicamente para geração tanto de biocombustíveis quanto de produtos químicos úteis. Estudos de conversão térmica de microalgas tem aparecido apenas em anos recentes, endereçando principalmente temperaturas entre 350 e 500 ºC. Neste estudo, a pirólise em temperaturas entre 500 e 900 ºC foi avaliada pelo interesse em se obter biocombustíveis e produtos químicos úteis. As classes de compostos obtidos a partir da pirólise isotérmica da microalga da espécie Chlorella vulgaris foram investigadas através de um sistema composto por um pirolisador conectado a um CG/MS. Dentre as classes identificadas, houve uma predominância de compostos aromáticos e nitrogenados, principalmente derivados de proteínas. Além disso, a maioria dos compostos identificados na corrente de produtos voláteis está presente em todas as temperaturas de reação estudadas. Os parâmetros cinéticos de energia de ativação aparente, constante de reação e ordem de reação foram determinados através de três modelos cinéticos conhecidos como K-A-S, Osawa e Freeman-Carroll, a partir de dados termogravimétricos. A energia de ativação determinada para Chlorella apresentou valores entre 60 e 206 kJ/mol, enquanto a ordem de reação teve como resultado valores entre segunda e décima ordem, de acordo com o modelo cinético. / The increasing need to obtain more sustainable sources of energy and raw materials has attracted attention to processes based on biomass conversion. Microalgae are particularly interesting because they do not compete with human food and they are easily adaptable to the growth medium. Besides, microalgae can be thermally processed to produce both biofuels and useful chemicals. Studies on thermal conversion of microalgae have appeared only in recent years, addressing mainly reaction temperatures between 350 and 500 °C. In this study, pyrolysis in temperatures ranging from 500 to 900 ºC have been investigated considering the interest in producing biofuels and useful chemical compounds. The classes of products obtained by isothermal pyrolysis of the Chlorella vulgaris microalgae have been investigated using a pyrolyzer directly connected to a GC/MS system. Among the classes identified, there was a predominance of aromatic and nitrogenous compounds, mainly protein derived compounds. In addition, most products identified in the volatile products stream are present in all reaction temperatures investigated. The kinetic parameters of apparent activation energy, reaction constant and order of reaction were determined through three kinetic models known as K-A-S, Osawa and Freeman-Carroll, calculated from thermogravimetric data. The activation energy for Chlorella presented values within the range of 60 and 206 kJ/mole, while the order of reaction resulted in values between second and tenth order, from the different methods.

La recherche visait à développer une technologie de biofilm à base de biofilms de microalgues pour le traitement des effluents riches en phosphore en vue de la production de produits de valeur secondaires, tels que la récupération du phosphore.Des recherches fondamentales ont été menées pour sélectionner des souches de microalgues efficaces en termes de traitement efficace des eaux usées et de production de biomasse algale, afin de mettre au point un procédé avec une faible addition d'électricité et de réactifs chimiques. Au total, 21 souches de microalgues ont été sélectionnées et sept souches ont été sélectionnées en fonction de leur taux de croissance. Le taux de croissance et l'efficacité d'élimination des éléments nutritifs de six souches ont été évalués dans différentes conditions de traitement des eaux usées synthétiques. Chlorella vulgaris ACUF_809 a montré une efficacité d'élimination du phosphate supérieure à celle des souches sélectionnées. Chlorella sp. ACUF_802 a également été caractérisé par l'effet des conditions limitant l'azote et le phosphore dans le milieu de croissance. Des expériences en laboratoire ont été effectuées pour évaluer les performances de formation de biofilm sur des tissus. L’activité photosynthétique, le pourcentage de colonisation et la croissance de la biomasse ont été étudiés pour deux Chlorella spp. Les différences dans les performances de formation de biofilm étaient observées entre les souches. Il a été constaté que l’analyse combinée de méthodes optiques non destructives est une méthode efficace pour surveiller les premières étapes du développement d’un biofilm. L'épaisseur moyenne du biofilm (106,37 ± 47 µm) a été mesurée. En outre, la distribution et la localisation des phosphates au cours du développement des biofilms C: pyrenoidosa ACUF_808 et C. vulgaris ACUF_809 ont été examinées au fil du temps, tout en préservant le contexte spatial. En utilisant des mesures physiologiques combinées, des techniques microscopiques, spectroscopiques et spectrométriques de masse ont été déterminées pour déterminer la croissance cellulaire, l'adhésion cellulaire, la performance physiologique, l'élimination des nutriments (phosphate et nitrate) et l'accumulation de phosphate visualisée au niveau des cellules dans les biofilms.La production de biomasse et l'efficacité d'élimination des nutriments par les microalgues Scenedesmus vacuolatus et C. vulgaris ACUF_809 à partir d'eaux usées synthétiques dans un nouveau photobioréacteur (PBR) de biofilm en laboratoire ont été étudiées. Le développement de biofilms sur du tissu de coton a été suivi dans le PBR dans des conditions semi-discontinues pendant 41 jours. S. vacuolatus ACUF_053 a obtenu l’élimination complète des éléments nutritifs et cinq étapes de développement du biofilm ont été identifiées: 1) fixation, 2) formation de biofilm, 3) maturation I, 4) sélection / adaptation et 5) maturation II. En revanche, le développement du biofilm de C. vulgaris ACUF_809 a montré une croissance de la biomasse plus régulière et une efficacité d'élimination constante. Cette recherche offre de nouvelles informations fondamentales dans le domaine de la formation de biofilms de microalgues pouvant être utilisées dans un plus large éventail d'applications scientifiques, notamment la possibilité d'un traitement couplé des eaux usées avec une récupération potentielle du phosphate / The aim of this Ph.D. research was to develop a bench scale microalgae biofilm technology for phosphorus rich effluents treatment toward the production of secondary valuable products, i.e. phosphorus recovery.Fundamental research was conducted to select efficient microalgae strains in terms of successful wastewater treatment and algal biomass production, in order to develop a process with low addition of electricity and chemical reagents. A total of 21 microalgae strains were screened and seven strains were selected according to their growth rate. The growth rate and nutrient removal efficiency of six strains were evaluated under different synthetic wastewater conditions, Chlorella vulgaris ACUF_809 showed superior efficiency of phosphate removal compared with the strains screened. Chlorella sp. ACUF_802 was characterized additionally by the effect of nitrogen and phosphorus limitating conditions in the growth medium. Laboratory scale experiments were done to evaluate the biofilm formation performance on textile fabrics. Photosynthetic activity, colonization percentage and biomass growth were investigated for two isolated Chlorella spp. Diffferences in biofilm formation performance were obswerved between the strains. It was found that combined analysis of non-destructive optical methods is an effective methodology for monitoring the early stages of biofilm development. Biofilm average thickness (106.37 ± 47 µm) was measured. Furthermore, as a promising valuable recovery product, phosphate distribution and localization during C: pyrenoidosa ACUF_808 and C. vulgaris ACUF_809 biofilms development were examined over time and keeping the spatial context unaltered. Using physiology measurements combined to advance microscopic, spectroscopic and mass spectrometric techniques were determined cellular growth, cell adhesion, physiological performance and nutrient (phosphate and nitrate) removal and visualized phosphate accumulation at single-cell level within the biofilms.Biomass production and nutrient removal efficiency by the microalgae Scenedesmus vacuolatus and C. vulgaris ACUF_809 from synthetic wastewater in a new laboratory configuration biofilm photobioreactor (PBR) was studied. Biofilm development on cotton fabric was followed in the PBR under semi-batch conditions during 41 days. Complete nutrient removal was obtained by S. vacuolatus ACUF_053 and five stages in the development of the biofilm were identified: 1) attachment, 2) biofilm formation, 3) maturation I, 4) selection/adaptation and 5) maturation II. In contrast, C. vulgaris ACUF_809 biofilm development showed a more regular biomass growth and constant removal efficiency. This research offers novel fundamental information in the field of microalgae biofilms formation with a scope for a wider range of scientific applications, including the possibility of coupled wastewater treatment with potential recovery of phosphate