Thèses sur le sujet « Tetraselmi »

The research described in thesis deals with the production of biofuels and fine chemicals from the green alga Tetraselmis suecica. Firstly, the identity of the strain received from the culture collection was confirmed using molecular techniques (18S rDNA sequencing) and electron microscopy. Secondly, a fully defined artificial seawater medium was developed to grow T. suecica and then the tolerance of this alga to salinity and pH changes was established. The neutral lipid (triacylglycerol) production was measured using Nile Red dye after stressing T. suecica cells with high salinity (up to 1 M NaCl) and pH values (pH 7 to 9). It was established that high salinity and high pH values tended to induce higher levels of triacylglycerol in the algal cells. Then fatty acid profiles of T. suecica cells were analyzed by gas chromatography–mass spectrometry (GC-MS) after direct transesterification with hydrochloric acid in methanol. Higher salinity grown cells showed higher levels of monounsaturated fatty acids, which are ideal for biodiesel production. The possibility of growing T. suecica on a larger scale was investigated using a 2 L airlift photobioreactor and the response to higher levels of CO2 was assessed in the airlift bioreactor. The effect of reusing the medium on the growth of T. suecica was examined with the aim of developing anintegrated algal biorefinery process using T. suecica as the feedstock.

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
Atualmente, devido ao aumento do número de pessoas que sofrem da doença celíaca, o mercado dos alimentos isentos de glúten tem vindo a expandir a oferta, no entanto, para além de não serem sensorialmente apelativos, o preço final ao consumidor é elevado. A ausência do glúten no pão representa um desafio tecnológico, pois é necessário recorrer a matérias primas alternativas e que mimetizem as propriedades elásticas assumidas pelo glúten. Em simultâneo, também tem crescido a procura por alimentos com ingredientes bioativos e as microalgas são reconhecidas como fonte de diversos compostos com impacto positivo na saúde humana. O principal objetivo deste trabalho foi o desenvolvimento de pães isentos de glúten com incorporação da alga Tetraselmis chuii. Esta é uma microalga verde unicelular com propriedades nutricionais interessantes para a saúde humana. No decorrer do trabalho foi desenvolvida uma formulação controlo à base de farinha de trigo sarraceno, farinha de arroz e amido de batata, utilizada como base para estudar o impacto da adição de teores de alga de 1%, 2% e 4% (m/m). Avaliou-se a textura e reologia das massas, o perfil de envelhecimento do pão, os parâmetros de cor da massa e do pão, a atividade da água e humidade do miolo dos pães, o volume final dos pães e as perdas durante a cozedura dos pães. A incorporação de biomassa microalgal de Tetraselmis chuii originou produtos com características tecnológicas dependentes do nível de incorporação de microalga. Para níveis de 1% e 2% (m/m) verifica-se uma desestabilização da estrutura desenvolvida pelo amido e o HPMC, obtendo-se pães mais compactos, mais firmes e com menor volume do que o pão controlo. Para 4% (m/m) de incorporação de Tetraselmis chuii, as proteínas da microalga em associação com o amido e o HPMC desenvolvem um outro tipo de estrutura, reforçando a viscoelasticidade da massa, dando origem a um pão com maior volume e menor firmeza. Verificou-se ainda que a incorporação de microalga tem um efeito positivo no envelhecimento do pão. A análise sensorial hedónica realizada a três das formulações – controlo, 1% e 4% (m/m) de microalga, recorrendo a um painel de 32 provadores não treinados, resultou na eleição da formulação com 1% (m/m) de microalga como a preferida do painel
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Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia / UL
As microalgas são ainda um recurso aquático em fase inicial de exploração. A composição em ácidos gordos, compostos fenólicos, perfil fenólico e bioatividades foram analisadas em três novas estirpes de microalgas: Tetraselmis sp. IMP3, Tetraselmis sp. CTP4 e Skeletonema sp. Foi também estudada a estabilidade de extratos de Skeletonema sp. não encapsulados e microencapsulados em inulina
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A praia de Itacoatiara, área de proteção ambiental, localizada no município no Niterói- RJ, tem importante contribuição para o turismo ecológico da região. Busca a certificação ambiental do programa Bandeira Azul e este trabalho buscou cumprir determinação de monitoramento ambiental como um dos critérios do programa. O objetivo foi avaliar a proliferação de microalgas marinhas como sensor da qualidade da balneabilidade diagnosticada através de um bioindicador, o fitoplâncton. As coletas foram realizadas em dois pontos nos meses de novembro de 2008 e de janeiro a março de 2009. Foram encontradas 52 unidades taxonômicas, sendo que 33 pertencem às diatomáceas (Bacillariophyceae), 15 dinoflagelados (Dinophyceae), 1 às cianobactéria (Cyanophyceae), 1 euglenofícea (Euglenophyceae), 1 crisofícea (Chrysophyceae) e 1 prasinofícea (Prasinophyceae) e fitoflagelados. A metodologia seguida foi de sedimentação em cubetas e analisadas em microscópio invertido. A praia de Itacoatiara está caracterizada por sofrer influência de ambientes adjacentes com massas dágua comprometidas promovendo proliferações algais monoespecíficas com densidades consideradas de Bloom. As espécies dominantes foram Tetraselmis sp (Prasinophyceae) considerada potencialmente nociva por apresentar contaminação visual e as Cianobactérias que podem produzir toxinas. Os parâmetros vento, ondulação e maré foram apontadas como os principais carreadores das massas dágua comprometidas até a região estudada. Por cumprir o objetivo geral, conclui-se que o fitoplâncton marinho pode ser utilizado como parâmetro para a caracterização e qualidade da balneabilidade de praias.
The Itacoatiara beach, area of environmental protection, in the municipality in Niterói, has an important contribution to the ecological tourism in the region. Search the environmental certification of the "Blue Flag" and this work seeks to achieve environmental monitoring determination as one of the criteria of the program. The objective was to evaluate the proliferation of marine microalgae. The quality of bathing was diagnosed by a bioindicator, the phytoplankton. The collections were made during the months from November 2008 to March 2009. 52 taxonomic units were found, with 33 belonging to the diatoms (Bacillariophyceae), 15 dinoflagellates (Dinophyceae), 1 cyanobacteria (Cyanophyceae), 1 Euglenophyceae, 1 Chrysophyceae and 1 Prasinophyceae and phytoflagellates. The methodology was based in sedimentation cuvette and analysis in inverted microscope. The beach is characterized by the Itacoatiara influence of environments adjacent to the water masses involved in promoting proliferation monospecific algae densities considered of "Bloom". The dominant species were Tetraselmis sp (Prasinophyceae) considered potentially harmful contamination by presenting visual and cyanobacteria that can produce toxins. The parameters wind, waves and tides have been identified as the main carriers of the water masses committed to the region studied. By meeting the general objective, we find that the marine phytoplankton can be used as parameter for the characterization and quality of balneability of beaches.

The distribution of photosystem I (PS I) and photosystem II (PS II) in a primitive green alga Tetraselmis subcordiformis, which belongs to Prasinophyceae and does not have grana in its chloroplast, was studied by immunoelectron microscopy. Two PS I antibodies were used: one against a PS I component of maize, the other against the 60 and 62 KDa PS I reaction centre proteins of the cyanobacterium Synechococcus elongatus. Both antibodies showed that 76-78% of the labelling is on the appressed thylakoid membranes and only 22-24% is located on the unappressed membranes. Use of antiserum against cp-47 of PS II from S. elongatus also gives 76% of the labelling on appressed thylakoid membranes and 24% on unappressed thylakoid membranes. Cytochemical detection of PS I activity by the photooxidation of 3,3$ sp prime$-diaminobenzidine and of PS II activity by the photoreduction of distyryl nitroblue tetrazolium chloride also revealed that PS I and PS II activities exist on both types of thylakoid membranes. Therefore, our results indicate that the distribution of PS I and PS II in green algae may differ from that in higher plants.

The effects of stress conditions on neutral lipid accumulation by the microalgae Botryococcus braunii, Tetraselmis suecica and Coccomyxa were investigated to assess their suitability for biodiesel production. The growth media (3N-BBM+V for B. braunii, and Coccomyxa and F/2 for T. suecica) were altered to impose salt stress (0.1 and 0.2 M NaCl for BBM medium) and up to 1.2 M NaCl for F/2 medium. Tetraselmis suecica and Coccomyxa were also cultivated under nitrogen depletion conditions (50%, 25% nitrogen and nitrogen free) to monitor growth and investigate neutral lipid accumulation. It was found that B. braunii grew very slowly despite using the relatively nutrient rich BBM medium. It was difficult to reach sufficient biomass to run experiments for B. braunii, but faster growth was achieved using a 2 litre fermenter. B. braunii had a total lipid content of 29% but only 3.64% were neutral (storage) lipids. This is much less than the 30% neutral lipid normally quoted as the minimum requirement for biodiesel production (Chisti, 2007). The B. braunii strain (CCAP 807/1) used failed to grow in the presence of 0.1 or 0.2 M NaCl. Tetraselmis suecica grew in the higher salinity media, but salt stress did not induce higher lipid accumulation. Nitrogen free medium did induce more neutral lipid accumulation in T. suecica even though the growth rate and final biomass level reached were decreased. Tetraselmis suecica had less than the 30% neutral lipid normally quoted as the minimum requirement for biodiesel production, but it is an excellent candidate for production of biodiesel based on the fatty acids produced which are principally C16:0 and C18:1. Neutral lipid accumulation increased rapidly with increasing nitrogen starvation from 25% nitrogen to nitrogen-free medium as Coccomyxa grew to reach stationary phase over four weeks. Nitrogen free medium induced more neutral lipid accumulation (31%) in Coccomyxa, which was the highest percentage found in the current work and meets the 30% neutral lipid figure suggested by Chisti et al. (2007).

A capacidade de ligação de prótons e metais por células vivas da microalga Tetraselmis gracilis (Kylin) butcher foi estudada por titulação potenciométrica e voltametria de redissolução anódica, respectivamente. Dados da titulação alcalimétrica obtidos com uma suspensão de microalga suspensa em NaCl 0,60 mol L-1 a 25,0 ± 0,1 ºC foram tratados por um modelo de distribuição discreta de sítios baseado na linearização das curvas de titulação por funções de Gran modificadas. A concentração total de sítios ionizáveis foi 3,4 x 10-3 mmol g-1, divididos em quatro classes de grupos com pKa 4,4; 5,2; 7,0 e 9,3, cujas abundâncias relativas foram 13, 5,8, 8,2 e 73%, respectivamente. Ligação de Cd(II) e Zn(II) foi estudada em água do mar (pH 8,2) por adições de soluções dos cátion metálicos em soluções simples ou misturas binárias. O cálculo das concentrações dos metais a partir dos dados voltamétricos considerou o agregado célula-metal tendo difusão muito menor do que a do íon livre. Capacidades de adsorção e o logaritmo das constantes de equilíbrio condicionais foram 7,9 ± 0,9 µmol g-1 e 6,9 ± 0,3 L g-1 para Cd(II) e 18,1 ± 0,4 µmol g-1 e 8,8 ± 0,2 L g-1 para Zn(II). Nas titulações com misturas binárias as capacidades de adsorção foram 0,129 ± 0 ,008 e 18,1 ± 0,4 µmol g-1 de Cd(II) e Zn(II), respectivamente, sugerindo que a ligação de Zn(II) inibe a de Cd(II). Entretanto, mesmo na presença de Zn(II) a superfície de alga possui sítios minoritários que se ligam a Cd(II) (log Kads = 7,7 ± 0,2), o que pode causar a bioacumulação desse íon metálico tóxico.
Proton and metal binding capacities of living cells of the microalgae Tetraselmis gracilis (Kylin) butcher were determined by potentiometric titration and anodic stripping voltammetry, respectively. Data of alkalimetric titration of the microalgae suspension obtained in 0.60 mol L-1 NaCl at 25.0 ± 0.1ºC was treated by a discrete site distribution model based on the linearization of the titration curve by modified Gran functions. A total of 3.4 x 10-3 mmol g-1 ionizable sites were determined, divided in four classes of groups with pKa 4.4, 5.2, 7.0 and 9.3, whose relative abundances were 13, 5.8, 8.2 and 73%, respectively. Binding of Cd(II) and Zn(II) was studied in seawater (pH 8.2) by additions of either single metallic species or binary mixtures. Computation of free metal concentrations from the voltammetric data considered the cell-metal aggregates with diffusion coefficient significantly lower than that of free metal ions. Adsorption capacities and the logarithm of the conditional equilibrium constants were 7.9 ± 0.9 µmol g-1 and 6.9 ± 0.3 L g-1 for Cd(II) and 18.1 ± 0.4 µmol g-1 and 8.8 ± 0.2 L g-1 for Zn(II). For titrations with binary mixtures of Cd(II) and Zn(II ) the adsorption capacity of Cd(II) were 0.129 ± 0.008 µmol g-1 and 18.1 ± 0.4 µmol g-1, respectively. The results suggest that binding of Zn(II) inhibits that of Cd(II). However, even in the presence of Zn(II), the alga surface has some minor sites that can bind Cd(II) (log Kads = 7.7 ± 0.2), a process that can lead to bioaccumulation of this toxic metal ion.

L'augmentation de la consommation du pétrole, principale source d'énergie fossile actuelle, utilisée en grande partie par le secteur du transport, fera en sorte que ses réserves risquent de diminuer dans le futur. Ainsi, la dépendance des pays consommateurs vis-à-vis des pays producteurs ne fera que s'accentuer. Par ailleurs, les transports sont considérés comme la première source d'émissions de gaz à effet de serre (GES) dont le dioxyde de carbone (CO2), gaz lié aux changements climatiques. Ces facteurs ainsi que l'instabilité des cours du pétrole ont incité les politiques et par conséquent les chercheurs à trouver, de façon urgente, des sources alternatives au pétrole. Le retour au biodiesel initié et utilisé, il y a plus d'un siècle, par Rudolph Diesel (1858-1913), s'avère une des solutions possibles. Ces dernières années, la plupart des biodiesels industriels sont produits à partir de l'huile (triglycérides) extraite de matières premières végétales (colza, tournesol, soja, etc.). Afm de changer leurs propriétés physico-chimiques pour les rendre similaires à celles du pétro-diesel, les triglycérides sont transestérifiés en esters alkyliques d'acides gras, qui peuvent être utilisés dans un moteur classique, sans modification. Sur le plan écologique, en plus de la capacité des plantes oléagineuses à réduire les émissions polluantes de GES en piégeant et en consommant notamment le CO2, l'utilisation du biodiesel réduit les émissions nettes de polluants. Or, une forte demande des plantes oléagineuses comme matières premières pour produire du biodiesel pourrait augmenter le prix des huiles qui sont également nécessaires à l'alimentation humaine. D'autre part, l'utilisation de telles huiles engendre des coûts de production de biodiesel. Afm de surmonter ces problèmes sociaux, économiques et environnementaux, l'utilisation des huiles usées et des microalgues comme matières premières s'avère la solution la plus plausible. Concernant les huiles usées, le rejet de ces résidus graisseux dans le réseau d'assainissement est un risque pour l'environnement et pour les installations de traitement des eaux. Ces huiles gênent le bon fonctionnement des stations d'épuration lorsqu'elles sont rejetées dans le réseau d'assainissement, ce qui entraîne un surcoût. Le déversement de ces huiles provoque de nombreuses nuisances à savoir l'obturation des canalisations, la difficulté de traitement des graisses en station d'épuration, la production d'odeurs nauséabondes et de gaz toxiques, la corrosion des canalisations et le déséquilibre de la faune et de la flore aquatique, d'où l'intérêt de les valoriser en biodiesel. Quant aux microalgues, leur valorisation en biocarburant se développe depuis plusieurs années. Ce sont des microorganismes très anciens et il en existerait au moins 100 000 espèces, dont une cinquantaine seulement sont bien connues parmi les 40 000 déjà étudiées. Il reste donc encore un très grand potentiel à explorer. Ces microorganismes capturent, concentrent et fixent l'énergie de la lumière dans la biomasse. Les procédés de production des microalgues sont variés. Certains d'entre eux permettent d'absorber les émissions de CO2 atmosphérique de divers procédés comme celles issues des cimenteries. La grande capacité des microalgues à épurer les effluents industriels, municipaux ou agricoles, peut aussi être avantageusement exploitée. Les microalgues présentent un potentiel de productivité pouvant dépasser dix fois les meilleures cultures agricoles conventionnelles. La production industrielle de microalgues se présente donc, de plus en plus, comme une solution durable intéressante. Les modes et les procédés de production ont évolué au cours des années. Le mode autotrophe, le plus répandu, développé avec l'énergie salaire, permet de convertir le CO2 en microalgue polyvalente. Les procédés autotrophes les plus étudiés sont constitués de divers types de bassins et de photobioréacteurs (PBR). Par ailleurs, les procédés hétérotrophes utilisent des bioréacteurs fermés alimentés d'effluents riches en hydrates de carbone. Les procédés hétérotrophes, indépendants des conditions climatiques, représentent donc un potentiel d'exploitation intéressant pour les zones nordiques. Le procédé mixotrophe consiste à cultiver des microalgues dans l'obscurité avec un apport de lumière variable au cours du temps. Cette étude entre dans le cadre de la production de biodiesel par valorisation d'une part des huiles de friture usées provenant du restaurant de l'Université de Sherbrooke et des huiles extraites de la microalgue Tetraselims sp. d'autre part. [symboles non conformes]

"I would put my money on one fundamental principle ... all life evolves by the differential survival of replicating entities" (Richard Dawkins) This study is aimed towards understanding the complexity in unicellular eukaryotic phytoplankton populations. For my research work I have chosen Tetraselmis (Prasinophyceae) as the test organism. Prasinophycean algae are considered as ancestors of all green algae and embryophytic land plants. Tetraselmis is a key transition organism in the phylogeny of green algae. The taxonomy and phylogeny of the genus was investigated using morphological, ultrastructural, flow cytometry and molecular cladistics analysis. Partial 18s rDNA sequences from fresh material and from gene bank were analysed. In this process a new species of Tetraselmis (T. indica) from salt pans of Goa, India has been reported. The high structural and molecular divergence observed in this species pointed towards the evolution in function, hence, T. indica was chosen as the test organism. T. indica inhabits the highly dynamic salt pan ecosystem and survives in wide fluctuations of salinity and environmental parameters. Studies at cellular level, where changes in cell physiology, biochemistry and optical properties were studied as a means of evaluating their function and response to alteration in the environment were carried out. Experiments suggested the existence of structural, physiological and metabolic differentiation in Tetraselmis as indicated by flow cytometric studies, differential absorption spectras and staining patterns. Further it was observed that an incipient differentiation exists in otherwise identical cells giving rise to a heterogenous population whose components exhibit differential survival and behaviour, which work in association for the existence of population. Investigation of the biology of a single cell and its lineage helped in deciphering the consequences and implications of heterogeneity and complexity in this unicellular eukaryote. To study how component cells of a heterogenous population achieve their own behaviour, the hierarchy of subcomponents within these component cells was explored. Studies suggested that the fate of different components is set at a very early stage of cell division and hence the division products of a single cell exhibit ultrastructural and metabolic differences. To consider the social interactions, naturally associated bacteria were inoculated with the axenic cultures of cells and the differential and marked influence of each bacterium on its growth dynamics was observed. This study highlights that interrogating the complete web of individual and social interactions is of importance in understanding the functionality and regulation of unicellular life.

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
Estima-se que 1% da população mundial e 3% da população nacional seja intolerante ao glúten. Estes doentes necessitam de uma dieta restrita de glúten (APC 2019). Simultaneamente, a população não celíaca tem demonstrado interesse na alimentação isenta de glúten, defendendo os seus beneficios a nível nutricional. Consequentemente, a procura pelos produtos isentos de glúten tem vindo a aumentar apesar destes serem considerados caros e com deficiências a nível sensorial. Como forma de melhorar a oferta de alimentos isentos de glúten, prezando-se pela inovação, vários ingredientes, como as microalgas, têm sido explorados. Estas são conhecidas pelo elevado valor proteico, constituição em lípidos, hidratos de carbono e compostos bioativos com poder antioxidante. O principal objetivo deste trabalho foi o estudo do impacto do pré-tratamento para a disrupção celular da biomassa microalgal no desenvolvimento de pães isentos de glúten com incorporação de Tetraselmis chuii. A disrupção celular é um processo que promove a libertação de compostos presentes no interior da parede celular. Para estudar a funcionalidade da microalga e o impacto da disrupção celular e do tratamento térmico subsequente, prepararam-se suspensões de Tetraselmis chuii a 10% (m/v) com diferentes níveis de disrupção celular, estudou-se o comportamento reológico, concentração de compostos fenólicos e compostos com atividade antioxidante, através dos métodos de Folin-Ciocalteu, DPPH e FRAP. Para os sistemas concentrados, massas e pães, avaliou-se a textura e reologia das massas, aspeto geral, textura e parâmetros de qualidade dos pães, cor das massas e pães, e concentração de compostos bioativos. Nos sistemas diluídos as suspensões apresentam uma alta capacidade antioxidante, apesar de a concentração de compostos fenólicos ser reduzida. A incorporação de biomassa microalgal em sistemas concentrados originou produtos com elevado poder antioxidante, promovido pelos níveis de disrupção elevados, para além de características tecnológicas desejáveis para utilização industrial, com reforço da estrutura devido à maior firmeza
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The push for alternatives to petroleum fuels has forced researchers to look for highly productive, renewable, non-food resources. The advantages of using microalgae instead of traditional oil crops for biofuel production include high oil yields, rapid reproductive rates, and versatile growing requirements. In order to reduce the cost of producing microalgae based biofuels, wastewater has been used as a nutrient source instead of specialized plant nutrients. The purpose of this study was to compare the relative effectiveness of different combinations of microalgae strain and dairy wastewater for increasing biomass. The methods for monitoring growth included optical density, cell counting, biomass estimation by chlorophyll-a, and volatile suspended solids. The analyses compared four concentrations of wastewater media as well as four strain treatments: Chlorella vulgaris, Tetraselmis sp., mixed freshwater culture and mixed saltwater culture. Optimum wastewater concentrations for microalgae growth were approximately 0% and 25% for most strain treatments. The results of the wastewater treatments concluded that dairy wastewater could serve as an effective nutrient substitute for plant food at concentrations approximately 25%. Chlorella vulgaris and Tetraselmis sp. prevailed over the mixed cultures for biomass production. Nitrate was the most limiting nutrient and exhibited the greatest reductions, sometimes in excess of 90%. The regression equations derived from the volatile suspended solids data achieved high R2 values and determined that total nitrogen, ammonium, and nitrate were significant in the model. In those equations, increasing either ammonium or nitrate yielded an increase in volatile suspended solids. With regards to comparing biomass quantification methods, the two most useful and reliable biomass quantification methods were optical density and volatile suspended solids.

Il futuro sarà caratterizzato da un trend demografico in aumento che, unito ad un continuo miglioramento degli stili di vita, e all’incremento del benessere sociale, porterà ad un innalzamento della domanda di beni di sussistenza, ed in particolare di generi alimentari, che si scontrerà con un’offerta sempre più limitata da un ecosistema sovrasfruttato. Ciò si può osservare chiaramente nel caso della pesca: secondo gli scenari di previsione elaborarti dalla Banca Mondiale, per il 2030, a livello globale, ci sarà una richiesta di 261 milioni di tonnellate solo di prodotti ittici; ma già al giorno d’oggi l’eccessivo ricorso alla pesca tradizionale sta impoverendo le risorse marine con un conseguente danno all’ecosistema ed alle specie che lo abitano. Per ovviare almeno in parte al problema, negli ultimi 50 anni è stato sempre più sviluppato il settore dell’acquacoltura. Sono moltissime le ricerche effettuate per aumentare le conoscenze in questo settore e proporre innovazioni per renderlo sempre più efficiente; ne è un esempio il progetto triennale AlgaSphera (PR.FEM.PPA201700.3 / PR.FEM.PPA201700) che ho potuto seguire per alcuni mesi. Il progetto è iniziato nel 2017 presso l’istituto andaluso di investigazione e formazione agraria ed ittica, IFAPA, de El Toruño, nella provincia di Cadice, in Spagna. L’obiettivo principale è ottenere microsfere capaci di conservare biomassa viva di microalghe per lunghi periodi di tempo, al fine di impiegarle in acquacoltura come alimento per la fase iniziale dell’alimentazione esogena di pesci marini. Tali particole sono state prodotte incapsulando, con l’utilizzo di un incapsulatore BUCHI B-390, una miscela di alginato e microalghe concentrate vive dei generi Tetraselmis e Diacronema. Sono state successivamente conservate e ne è stata monitorata la vitalità nel corso dei mesi attraverso analisi con colorante vitale Blu di Evans, analisi della fluorescenza, della capacità di crescita e dei nutrienti.

Advanced oxidation technology (AOT) has been used to destroy microorganisms in ballast water by breaking down the cell membranes. The primary objective of this study was to determine the effects of a ballast water treatment system that uses a combination of UV-C (λ=254 nm), Vacuum-UV (VUV; λ=185 nm) and photocatalytic titanium dioxide (TiO2) on a freshwater algae, Pseudokirchneriella subcapitata, and a marine algae, Tetraselmis suecica. The coupling of a semiconductor, such as TiO2, with a UV source is known as an advanced oxidative technology (AOT). To test the effects of TiO2 and wave length on algae, dose-response experiments were conducted to determine the species median lethal dose (LC50) for each of the following treatments: UV-light emitted at 254 nm (UVλ=254 nm), UV-light emitted at 254 nm in the presence of TiO2 (AOTλ=254 nm), and UV-light emitted at λ=254 nm (90 %) and 185 nm (90 %) in the presence of TiO2 (AOTλ=185 + 254 nm). In both species, TiO2 significantly increased mortality, most likely due to the biologically harmful radicals generated at the TiO2 surface. The addition of the 185 nm wavelength significantly increased cell mortality in P. subcapitata, but not in T. suecica. Across all three treatments, P. subcapitata was more sensitive than T. suecica. The secondary purpose of this study was to assess the applicability of ImageJ, an image analysis software, for highthroughput data to analyze the effectiveness of ballast water treatment. ImageJ has been used to rapidly and accurately perform cell Live/Dead analysis; however, several hurdles were identified.

La capacite qu'ont les microalgues d'accumuler les metaux traces est bien etablie. Toutefois, les mecanismes de bioaccumulation et de detoxification demeurent largement inconnus, notamment dans le cas du cadmium. Pour la comprehension de ces phenomenes, une etude ultrastructurale de skeletonema costatum et de tetraselmis suecica contaminees par le cadmium et/ou le cuivre, en condition chronique ou aigue a ete entreprise. Il en ressort globalement, que le cuivre, metal le plus toxique pour les deux microalgues, induit les plus fortes modifications structurales. Pour les deux metaux, le developpement d'organites de stockage est en relation avec les doses et les conditions de contamination. La distribution subcellulaire des polluants et des metabolites, impliques dans la bioaccumulation, a ete recherchee par les techniques analytiques de la microscopie electronique: edxs et eels. Cette derniere technique a permis de quantifier les concentrations de polluants ainsi que d'autres elements accumules dans les organites de stockage. La forme chimique de stockage a ete egalement recherchee par l'eels. Chez les deux microalgues, le cadmium est stocke sous forme oxydee cd#2#+, alors que le cuivre peut exister sous forme cu#2#+ et/ou cu#+. L'etroite correlation entre les concentrations de l'azote et du soufre et celle des polluants laisse supposer que des molecules de type phytochelatines peuvent etre impliquees dans les mecanismes de detoxification. Les metaux faiblement lies dans les vesicules de stockage sont susceptibles d'etre transferes le long des chaines trophiques. Aussi, la contamination par voie alimentaire de l'huitre (crassostreagigas) a ete entreprise. L'etude microanalytique (eels) a permis de comparer les voies d'entree et la forme de stockage des polluants dans le cas de la contamination directe ou alimentaire. Il en ressort que l'apport particulaire est aussi important que le transfert metallique par l'eau et que les formes de stockage des polluants sont equivalentes dans les deux cas du transfert

Algae are a promising new source of oil for biodiesel. They are aquatic organisms that do not require cropland, and they can produce many useful side-products for bioenergy, aquaculture, and nutraceutical production. To be cost-effective, algae need high and reliable oil productivities; however, there is still a great deal to learn about the effects of culturing conditions on algae growth rates and lipid production. These culturing conditions include light intensity, gas flow, use of CO₂, and culture volume. An extensive database of published research on algae growth rates and lipid contents under a wide variety of environmental conditions was prepared. By graphing data from 116 publications on 132 microalgae species, several key trends were identified relating to culturing parameters and algae biomass and lipid production. In addition, data from 131 publications on 128 microalgae species were graphed to look at presence of flagella, nutrient limitation, lipid productivity, and productivity tradeoffs. Moreover, cell size information was gathered for 146 species. The interactions between culture variables are complex, so it is difficult to quantify the degree to which each culture variable affects algae growth rates and lipid production. Therefore, several multivariate analyses were performed to generate a set of general and simple predictive models to assess specific growth rates, maximum lipid contents, and volumetric lipid productivities. These models were used to determine which culture parameters were significant predictors of algae growth rates and lipid production, and the contribution of each environmental parameter was quantified. In addition to models for algae in general, genera specific models were prepared for Chlorella, Isochrysis, Nannochloropsis, Phaeodactylum, and Tetraselmis. These models show high predictive capabilities, and they greatly extend the range of species-specific multiple regression models available. Furthermore, one Tetraselmis model was validated using Tetraselmis impellucida growth experiments in a large novel photobioreactor.

L’attività di ricerca svolta ha avuto come obbiettivo generale la coltura e la produzione di biomasse di microalghe marine selezionate e di buona qualità nutrizionale e il loro impiego come materia prima per la parziale sostituzione della farina e dell’olio di pesce nei mangimi per acquacoltura. Tutto questo al fine di creare un alimento innovativo perfettamente rispondente ai fabbisogni nutrizionali della spigola e di altre specie ittiche di pregio ed in linea con un modello di allevamento eco-sostenibile che privilegia l’impiego di sostanze e alimenti di origine naturale.

碩士
大葉大學
環境工程學系碩士班
96
With the increase of CO2 in the atmosphere, the effect towards the earth is becoming more and more serious. Among all, the most specific effect is Global Warming. Nowadays, one potential method is to fix the CO2 with and algal fixation algae by the light energy. By integrating mitigation the alkaline absorption process for industrial CO2 transaction, the mitigation of CO2 emission can be achieved effectively. In this work, solution of sodium bicarbonate was used to simulate the absorption dution from a scrubber in which carbon dioxide was absorbed by the solution of sodium bicarbonate. Bicarbonate ion in medium is the only carbon source for the growth of Tetraselmis chui. The effects of light intensity and concentration of sodium bicarbonate on the growth of Tetraselmis chui were studied in batch cultures. By batch development method with the factor control of inorganic carbon consistency and the light strength, the growing effect of the Tetraselmis chui were discussed as follows: The concentration of NaHCO3was changed from 0 g/L to 3.5 g/L for cultivation Tetraselmis chui. Above 2.0g/L, it showed no significant differences on growth rate but the increase of growing period. The variations of dissolved oxygen and pH value are not specific till the mid-term of the whole experiment. The significant effects of alkalinity are mainly on the length and the max value of growing period. With the increase in alkalinity, the dissolved oxygen and the PH value increased. With the increase in alkalinity, the amount of carbonate ion absorbed by Tetraselmis chui was increased. Furthermore, the growth period of Tetraselmis chui was significantly increased. The other cultivation conditions are: light intensities 7500 Lux and 15000 Lux; the medium is of the modified Walne’s medium. The compare of Light strength, which influenced the raising of Tetraselmis chui, is between 7500 Lux and 15000 Lux. The work of this research showed that the light intensity of 15000 Lux is better than 7500 Lux. The significant increasing was showed in the parts of growth rate, period of growing, pH value and the dissolved oxygen.

碩士
大葉大學
環境工程學系碩士班
96
In this study, the effects of light intensity, concentration of sodium bicarbonate and dilution rate on the growth of Tetraselmis chui were investigated in continuous cultivation using a single-tank photobioreactor. Solution of sodium bicarbonate was used to simulate the absorption solution from a scrubber in which carbon dioxide was absorbed by aqueous solution of sodium hydroxide. Bicarbonate ion in medium is the only carbon source for the growth of Tetraselmis chui. It was shown that change in concentration of sodium bicarbonate in medium had no significant effect on the growth of Tetraselmis chui in continuous cultures with concentrations of 0.5, 1, 2 and 3 sodium bicarbonate g/L . The culture pH was lower with higher concentration of sodium bicarbonate. During the batch mode of each continuous culture, microalgae grew fast and the biomass concentration was higher with higher light intensity. However, the biomass concentration became lower in continuous culture than that in batch culture due to the dilution of medium. The higher specific growth rate occurred at higher dilution rate, while the biomass concentration and pH were lower with higher dilution rate.

碩士
國立臺灣海洋大學
生物科技研究所
98
To investigate the biotransformation speciation of arsenic through Tetraselmis chui. in marine hydrosphere, the simulated arsenate concentration about 2 μg/L was applied to incubate Tetraselmis chui in phosphate concentration 0.2 mM and 2 mM respectively. The qualitative and quantitative of arsenic species in medium were measured by hydride generation-packed cold finger-atomic fluorescence spectrometer (HG-PCF-AFS) hyphenated system and arsenic species in Tetraselmis chui were measured by ion chromatography-ultraviolet digestion-hydride generation- atomic fluorescence spectrometer (IC-UV-HG-AFS) hyphenated system. The Tetraselmis chui and medium total arsenic mass were measured by graphite furnace atomic absorption spectrometer (GFAAS). For 0.2 μM phosphate incubation experiment, the phosphate concentration in medium is below the detection limit at day 6, and the growth of Tetraselmis chui cell is blurred due to the limit of phosphate. For experiment of 2 μM phosphate, the algal abundance showed log phase from day 2 to day 6 and after day 6 is stationary phase. The phosphate concentration decreased to 0.16 μM from day 2 to day 8 and then gradually increased to 0.61 μM at day 14. In 2 μM phosphate concentration experiment, only arsenite (AsIII) and arsenate (AsV) were detected in medium, and AsV decreased but AsIII increased from day 2 to day 4. The total arsenic were 2.59 μg/L at day 2 and then maintained in a range of 0.10-0.15 μg/L from day 6 to day 14, representing uptaked and accumulated arsenate by Tetraselmis chui. The measurements arsenic species in Tetraselmis chui showed the total arsenic concentration maintain in a range of 10-15 mg/kg and about 12~26% are water soluble arsenic species. In these water soluble arsenic species, AsIII contributed 0-44%, AsV contributed 5-17%. DMA were presented only at day 4 and day 8, and the concentrations were 0.34 and 0.49 mg/kg respectively. Arsenic cycle is very complex and need further study.

Tetraselmis sp. CTP4 was selected from a bioprospection screening as a promising candidate for industrial cultivation and exploitation of different biotechnological applications. At lab-scale, several experiments revealed that this strain is highly robust to environmental conditions, has the ability to accumulate significant amounts of lipids under nitrogen depletion, displays also high growth and sedimentation rates. In collaboration with Allmicroalgae (Algafarm, Secil, Leiria, Portugal), experiments at industrial-scale in 100-m3 tubular photobioreactors showed that this strain is able to attain promising areal productivities, remaining a monoalgal culture throughout the whole trial. Thereafter, a low-cost pilot-scale harvesting system enabled the recovery of 97% of the total biomass by natural sedimentation, reducing the harvesting costs by 93%. Biochemical characterization of industrially produced biomass revealed a high content of proteins and dietary fibres as well as interesting levels of chlorophyll, carotenoids and vitamins, whereas microbial pathogens and contaminants analysed were absent from the biomass. Later, upon the development of a biorefinery platform, the wet biomass was extracted with ethanol and fractionated using a liquid-liquid triphasic system, leading to four different streams: non-polar (NP), colloidal (CP) and water (WP) phases as well as the residual biomass (RB) leftover of the ethanolic extraction. The CP was characterized as a source of high value molecules, while the NP, WP and RB were successfully upgraded into biodiesel, bioethanol and biogas, respectively. The RB was also tested as an ingredient for juvenile seabream, showing to be a promising substitute to replace soybean meal in aquafeeds. Overall, Tetraselmis sp. CTP4 was successfully produced at industrial scale, a low-cost harvesting system was established, and the produced biomass had a promising composition suitable for prospective nutritional applications. In addition, the biorefinery approach implemented led to the production of different streams that were effectively upgraded into different bioproducts, namely biofuels and aquaculture feed.
No âmbito de um esforço de bioprospeção de novas microalgas para desenvolvimento biotecnológico realizado pelo grupo MarBiotech (Centro de Ciências do Mar, Universidade do Algarve), a estirpe Tetraselmis sp. CTP4 foi selecionada para o cultivo de biomassa à escala industrial e exploração de diferentes aplicações biotecnológicas. À escala laboratorial, ensaios preliminares revelaram que esta estirpe apresenta elevada taxa de crescimento e robustez para tolerar diversas condições ambientais. Adicionalmente, esta estirpe tem a capacidade de acumular quantidades significativas de lípidos, que podem chegar a 33% do peso seco da biomassa produzida, quando as culturas são submetidas a limitação de azoto no meio de crescimento. Estes ensaios revelaram também que a Tetraselmis sp. CTP4 apresenta uma composição lipídica muito interessante, e que após a conversão da fração de lípidos em biodiesel, as propriedades do biocombustível produzido estão dentro dos parâmetros regulados pelas normativas europeia e americana. Dado o elevado potencial desta microalga, foi estabelecida uma colaboração com a Allmicroalgae (Algafarm, grupo Secil, Leiria, Portugal), a maior unidade de produção de microalgas em sistemas fechados da Europa, para avaliar o crescimento das culturas desta estirpe em condições exteriores, utilizando fotobiorreatores tubulares industriais com um volume de 100 m3. Os ensaios realizados no exterior mostraram que foram necessárias oito semanas para realizar o aumento de escala de uma placa de agar até aos reatores de produção industrial. Durante o aumento de escala, realizou-se a otimização da produção em sistemas tubulares à escala piloto. Esta otimização mostrou melhores produtividades de biomassa quando a velocidade de cultura se encontra entre 0,65 a 1,35 m/s e com o valor de pH de 8,0 para injeção de CO2 na cultura. À escala industrial, verificou-se uma adaptação imediata das culturas aos sistemas de produção tubulares com produtividades areais muito promissoras (10-20 g/m2/d) e elevada eficiência fotossintética (3,5% da irradiância solar total), sendo possível manter uma cultura monoalgal durante todo o período de produção (60 dias). A sequestração de CO2 foi seguida no fotobiorreator de 100 m3, revelando uma eficiência média de mitigação de CO2 de 65% e uma relação de biomassa/carbono de 1,8. Posteriormente, foi desenvolvido um sistema de colheita piloto de baixo custo da biomassa de Tetraselmis sp. CTP4, nas instalações da Algafarm. Para este fim, adaptou-se um tanque cilindro-cónico para recolher a biomassa através da sedimentação natural das células sem custos energéticos. Os ensaios mostraram que após se introduzir a cultura no tanque, as células conseguem sedimentar na parte inferior do tanque após 24 horas, sendo possível recolher o meio de cultura pelas entradas laterais do sistema. Através do processo desenvolvido é possível recuperar 97% da biomassa total por sedimentação natural, sendo que se perdem apenas 3% com a remoção do meio de cultura do sistema. Usando esta abordagem, 93% do volume total da cultura é recuperado do tanque, com um peso seco de 0,07 g/L, o que representa uma redução muito significativa dos custos associados à colheita da biomassa. A restante cultura (7%) é recuperada na forma de uma cultura concentrada e pasta húmida de microalgas com pesos secos aproximados de 20 e 273 g/L, respetivamente. Na fase seguinte, pretendeu-se avaliar o potencial nutricional da biomassa de Tetraselmis sp. CTP4 produzida em fotobiorreatores industriais (100 m3). Para este fim, realizou-se uma avaliação minuciosa da composição bioquímica, microbiológica e toxicológica da biomassa. As análises efetuadas mostraram que a biomassa contém elevadas quantidades de proteína (31,2 g/100 g), fibras alimentares (24,6 g/100 g), glícidos digestíveis (18,1 g/100 g) e cinzas (15,2 g/100 g), mas com baixo conteúdo lipídico (7,04 g/100 g). A biomassa apresentou ainda níveis interessantes de clorofila (3,5 g/100 g), carotenóides (0,61 g/100 g) e vitaminas (por exemplo, 79,2 mg de ácido ascórbico/100 g) e atividade antioxidante. Por outro lado, bactérias patogénicas, metais pesados, cianotoxinas, micotoxinas, hidrocarbonetos aromáticos policíclicos e pesticidas não foram detetados na biomassa produzida. De um modo geral, a biomassa produzida tem uma composição promissora para aplicações nutricionais em humanos e animais. Subsequentemente, pretendeu-se desenvolver um novo método de processamento da biomassa tendo em conta o conceito de biorrefinaria, de modo a rentabilizar ao máximo todos os componentes bioquímicos presentes na biomassa de Tetraselmis sp. CTP4, para produzir diferentes bioprodutos. Neste contexto, realizou-se uma extração com etanol diretamente da biomassa húmida e o extrato resultante foi fracionado usando um sistema trifásico líquido-líquido (LTPS). No final deste processo, a partir da biomassa, obtiveram-se 4 frações distintas, nomeadamente as frações não polar (NP), coloidal (CP) e aquosa (WP), obtidas a partir do extrato etanólico, e a biomassa residual (RB) remanescente da extração etanólica. A fração CP foi caracterizada como fonte de moléculas de valor acrescentado, devido à presença de fosfolípidos e carotenóides, que apresentam uma elevada aplicabilidade para diferentes indústrias. As frações NP, WP e RB foram convertidas com sucesso em diferentes biocombustíveis, nomeadamente, biodiesel, bioetanol e biogás, respetivamente. No final desta dissertação, foi ainda realizado um ensaio em colaboração com a Sparos Lda. para testar a biomassa residual como um ingrediente para rações de aquacultura. Neste contexto, foi realizado um ensaio para avaliar o efeito de uma incorporação de 10% de biomassa residual de Tetraselmis sp. CTP4, em substituição da farinha de soja em juvenis de dourada (Sparus aurata). O ensaio foi realizado durante 61 dias e mostrou que os critérios gerais de desempenho (peso corporal final, índice de crescimento diário, taxa de conversão alimentar e taxa de eficiência proteica), composição corporal total e retenção de nutrientes não foram significativamente afetados pela introdução da biomassa residual. No entanto, a dieta com biomassa residual apresentou valores significativamente superiores nos coeficientes de digestibilidade aparente (ADC) de proteína, energia e fósforo, comparativamente à dieta com farinha de soja. No final, um teste de confinamento agudo mostrou uma resposta de cortisol significativamente menor nos peixes alimentados com a dieta com biomassa residual (120 ± 23 ng/mL) do que naqueles alimentados com a dieta com farinha de soja (160 ± 33 ng/mL). Os resultados gerais mostraram que a biomassa residual de Tetraselmis sp. CTP4 pode reduzir as elevadas necessidades de farinha de soja em alimentos para a aquacultura como atualmente se verifica. Em conclusão, a estirpe Tetraselmis sp. CTP4, foi produzida com sucesso à escala industrial, o sistema de recolha de biomassa de baixo custo foi efetivamente estabelecido e a biomassa produzida revelou um alto potencial para aplicações nutricionais. Além disso, a metodologia desenvolvida para o processamento da biomassa tendo em conta o conceito de biorrefinaria, permitiu a produção de diferentes frações que foram posteriormente convertidas em diferentes bioprodutos, nomeadamente biocombustíveis e rações para aquacultura.
The author acknowledges the Portuguese Foundation for Science and Technology (FCT) for funding the PhD fellowship (Grant SFRH/BD/105541/2014)

碩士
國立嘉義大學
水產生物學系研究所
94
Abstract The aim of this study approaches the feasibility of using microalgae to uptake NH4+ in water. There are four parts in this study: the first is to approach the effect of temperature on growth rate and NH4+-uptake rate of four microalgae; the second is to approach the effect of culture media, algal growth stages and inoculated algal concentrations of algae on the growth of Tetraselmis chui; the third is to approach the effect of light-path length of algal incubator, light intensity, aerial intensity and convention rate on growth rate and NH4+-uptake rate of Tetraselmis chui; the fourth is to approach the feasibility of the reuse of pond water whose NH4+ is uptake by microalgae, then filtrated by hard clam. The temperature for maximal growth rate and NH4+-uptake rate of Isochorysis, Tetraselmis, and Nannochloropsis is 25℃, while 35℃ is for Chaetoceros. At 25℃, the daily biomass productions per liter of Isochrysis, Nannochloropsis, Tetraselmis and Chaetoceros are 0.043,0.037,0.041 and 0.085 g dry weight, respectively. Daily NH4+-uptake per liter of these algae is 2.99, 4.27, 4.26 and 9.41 mg NH4+-N, respectively. Therefore, Tetraselmis has the highest ability in NH4+-uptake among these four microalgae. Inoculation with different growth stages of Tetraselmis chui affects their nutritional salt requirement. The addition of minor elements affects growth rate of Tetraselmis chui in mid-logarithmic phase insignificantly, while they increase growth rate of the algae in early-stationary phase. The addition of NH4+ increases the growth rate of algae in mid-logarithmic phase significantly. During 5-day culture, pHs in culture with NH4+ and NO3- as nitrogen sources are decreased and unchanged, respectively. Inoculated concentrations of algae affect growth rate and production of Tetraselmis chui in mid-logarithmic phase. The growth rate of algae with inoculated concentrations of 8 and 16 ×104 cells/ml reaches the highest. However, the production of algae with inoculated concentrations of 16 and 41 ×104 cells/ml is maximal. The growth rate and NH4+-uptake of Tetraselmis chui in the condition of light-path length of 8㎝, light intensity of 240 μE/m2.s and aerial intensity of 7 L/min are reach the highest in this experiment. They are 1.55 per day and 7.9 mg NH4+-N /L.day, respectively. In the growth experiment of tilapia, the control group (1) only proceeds one week due to a high ammonia-N in the water, while the control group (2) and the treatment group proceed three weeks. No significant difference in the growth rate and FCR of tilapia, and in ammonia-N in the water between the control group (2) and the treatment group was found. This indicates that the water exchange could be replaced by the reused water treated with algae and hard clam. In addition to saving of 340 L of artificial sea water, 340 L of algae water (OD750 nm 0.35) for clam food is produced in this study.

碩士
大葉大學
環境工程學系碩士班
95
Global warning has become an international issue. Emission of carbon dioxide (CO2) by combustion of fossil fuel has been considered the main cause of the global warning. Integration of alkaline absorption and algae cultivation has been proposed by researchers to remove carbon dioxide from flue gas. Absorption of carbon dioxide by alkaline solution results in high concentration of NaHCO3 which can be used as the carbon source to cultivate microalgae. Effects of NaHCO3¬ concentration, light intensity and the dilution rate on the behavior of continuous cultivation of Tetraselmis chui in a dual-tank photobioreactor using a modified Walne medium were investigated in this study. For each culture, pH values, dissolved oxygen (DO) concentrations, optical density (OD), biomass, chlorophyll, CO32- and HCO3- concentrations were measured. Specific growth rate, specific respiration rate, CO2 fixation rate and CO2 fixation efficiency were determined. Biomass, chlorophyll concentration and CO2 fixation rate increased with the increase in NaHCO3 concentration. The highest biomass concentrations in light reactor and in dark reactor were 0.23 g l-1 and 0.15 g l-1, respectively. The highest chlorophyll concentrations in light reactor and dark reactor were 3.27 g l-1 and 2.9 g l-1, respectively. Under variation of NaHCO¬3 concentration from 0.02 to 0.06 mol l-1, biomass productivity was fastest at 0.06 mol l-1 of NaHCO3 because the highest specific growth rate of 0.063 h-1 was found at 0.06 mol l-1 of NaHCO3. At steady state, among four different NaHCO3 concentrations, the highest CO2 fixation rate of 0.04 g h-1 was found at 0.06 mol l-1 of NaHCO3, but the highest CO2 fixation efficiency of 26% obtained at 0.04 mol l-1 of NaHCO3. With different light intensities in range of 10000~30000 Lux, the highest biomass concentrations were found at 30000 Lux in both light reactor and dark reactor were 0.21 g l-1 and 0.13 g l-1, respectively. The ratio of the highest biomass concentration to the lowest one was 1.9 : 1 for light reactor and 1.6 :1 for dark reactor. Both the highest specific growth rate of 0.065 h-1 and the highest specific respiration rate of 0.044 h-1 were obtained at 30000 Lux. Consequently, biomass productivity in light reactor and oxygen utilization in dark reactor of T.chui were fastest at 30000 Lux. The highest chlorophyll concentrations in light reactor and in dark reactor were respectively 2.79 g l-1 and 2.31 g l-1 which obtained at 30000 Lux. The highest CO2 fixation rate of 0.052 g h-1 and the highest CO2¬ fixation efficiency of 32.63% obtained at 30000 Lux by comparison with those at 10000 Lux and 20000 Lux. Variation of dilution rate from 0.03 to 0.16 h-1 indicated that the highest biomass concentrations obtained at dilution rate of 0.03 h-1 for both light reactor (0.24 g l-1) and dark reactor (0.20 g l-1). Among different dilution rates, the highest specific growth rate of 0.10 h-1 was found at 0.16 h-1. Chlorophyll concentration decreased with the increase in dilution rate. The highest chlorophyll concentrations were 8.82 g l-1 in light reactor and 6.83 g l-1 in dark reactor. In range of dilution rate from 0.03 h-1 to 0.16 h-1, the highest CO2 fixation rate of 0.09 g h-1 and the highest CO2 fixation efficiency of 23.1% obtained at 0.10 h-1 of dilution rate.

碩士
大葉大學
環境工程學系碩士班
101
In this study, light indensity distribution in a cylindrical photobioreactor was examined for cultures of Tetraselmis Chui by measuring light intensity in situ in the photobioreactor. Simulation for effects of light intensity, biomass concentration and radias of cylindrical photobioreactor on growth were investigated using a Haldane growth model for cultures of Tetraselmis Chui. Major events as following： （1）Biomass concentration and radius of a cylindrical photobioreactor affect light intensity inside the reactor. It was shown that Beer’s law could be applied to the attenuation of light intensity for cultures of Tetraselmis Chui. The absorption coefficient of light intensity ε was 0.663 L / g．cm obtained from results.Light intensity was hardly affected by biomass concentration as the optical density of culture below 0.5. （2）The growth rate of Tetraselmis Chui increased with the increase in light intensity in the range from 2000 to 5000 Lux. Growth inhibition was observed as the light intensity greater than 20000 Lux. The growth rate became saturated as the light intensity close to 10000 Lux. Key Words : Tetraselmis Chui, photobioreactor, light intensity distribution, Beer’s law

碩士
大葉大學
環境工程學系碩士班
94
Continuous cultivations in a light/dark dual-tank photobioreactor were conducted to study the effects of culture conditions on the growth of Tetraselmis chui using sodium bicarbonates as the carbon sources. The solution of sodium bicarbonate was used to simulate the alkaline solution after absorbing carbon dixide. The main results and conclusion are as the following： (1) The effects of the concentrations of sodium bicarbonate on the growth of Tetraselmis chui During the continuous cultivation, the biomass concentration of Tetraselmis chui was almost unaffected by changing the concentration of sodium bicarbonate in the feed tank from 3g/L to 10g/L. The biomass concentration and the specific growth rate were 0.245g/L and 0.053h-1, respectively, in the light tank and those were 0.174g/L and 0.039 h-1 in the dark tank. The pH in culture was lower with the higher concentration of sodium bicarbonate in the feed tank. (2) The effects of light intensities on the growth of Tetraselmis chui It was shown that the biomass concentration of Tetraselmis chui was unaffected as the light intensity increased from 30000Lux to 50000Lux.The light intensity of 30000Lux was identified in the light saturation region. Further cultures were conducted at the intensity of 30000Lux. (3) The effects of dilution rates on the growth of Tetraselmis chui The effects of dilution rates on the growth of Tetraselmis chui were performed with the dilution rate in the range from 0.033 to 0.099h-1.The specific growth rate in the light tank and the specific respiration rate in the dark tank increased as the dilution rate increased, while the pH of the culture decreased. However, the relationship between the specific growth rate and the specific respiration rate was not a linear one. (4) The effects of aeration on the growth of Tetraselmis chui Aeration in the continuous cultivations of Tetraselmis chui not only lowered the dissolved oxygen, but also the biomass concentration. Aeration had a negative effect on the growth of Tetraselmis chui.

The interactions occurring in the phycosphere between algae and bacteria are of different kinds and may influence the growth of microalgae. In the past few years, the growing interest on microalgal industrial and biotechnological applications hijacked the attention of the scientific community on the study of microalgae-bacteria interactions. A deeper knowledge of the composition of the microbiota associated to microalgae and on the mechanisms ruling microalgae-bacteria interactions may improve the current mass-culture techniques and enhance the productivity. Tetraselmis suecica is a green marine alga with several biotechnological applications. It is mainly used for aquaculture and cosmetic applications and for the production of bioactive compounds. Nevertheless, still little is known about the microbiota associated to T. suecica and its effect on the microalga growth, In this thesis the bacterial community associated with T. suecica (strain F&M M33) cultures, grown in different conditions, was described through a metagenomic approach, to evaluate the presence of a stably associated bacterial community. NGS metagenetic analysis were performed on four T. suecica cultures, derived from the same original culture but separated for years, and kept under different growing conditions. Despite the different characteristics of the cultures (seasons and cultures size), a “core” bacterial community was identified, accounting for the large part of the total bacterial community. Among the “core” OTUs it was possible to identify 7 different genera and at least 13 different families. However the relative abundance of the taxa forming the “core” community varied strongly, indicating that growing conditions and/or external contaminations influence the relative abundance of these microorganisms. Moreover, the isolation of strains belonging to the “core” community opened an interesting chance to evaluate the effects of these bacteria on T. suecica growth. The understanding of which molecules are involved in the interactions is a fundamental part in the study of microalgal-bacteria interaction. The associated bacterial population, release several compounds which may act as microalgal growth promoting features (MPG) affecting different aspects of microalgae such the composition in terms of lipids, pigments or cell dimensions. However, to date only few studies have phenotypically characterized bacteria associated with microalgae for the selection of microalgal growth-promoting bacteria. Therefore, in this thesis 200 bacterial strains were isolated from different cultures of Tetraselmis suecica, identified through 16S rDNA sequencing and phenotypically characterized for MPG traits. 46 strains were been identified as producers of MPG compounds: either indol-3-acetic acid (IAA) and/or siderophores. 22 isolates were tested in co-cultures with the axenic microalga to their effects on T. suecica growth: 4 of them showed a positive effect on microalgal growth. In particular two strains: Vitellibacter sp. and Sphyngopyxis flavimaris were able to enhance considerably the growth of T. suecica, in the condition tested. To deepen the knowledge of the molecules involved in the interactions, the supernatants T. suecica cultures, axenic and in co-culture with Vitellibacter sp. or Sphyngopyxis flavimaris, were analyzed with a non-targeted metabolomics approach. The characterization of the exo-metabolome of T. suecica, was described for the first time. It was possible to identify culture-characteristics compounds and several compounds involved in the metabolism of vitamins. It was observed that Sphyngopyxis flavimaris produce IAA also in co-culture conditions.

碩士
大葉大學
環境工程學系碩士班
94
The increase of the concentration of carbon dioxide in the atmosphere has been considered as the main cause of global warming. Fixation of CO2 using photosynthesis by microalgae is a kind of potential approach. The study aims to cultivate Tetraselmis Chui using bicarbonate ion as carbon source which is formed by alkaline absorption. Effects of culture conditions on the growth of Tetraselmis Chui were performed in batch cultures. The main results and conclusion as the following: 1. Effects of the concentrations of sodium bicarbonate on the growth of Tetraselmis Chui. Batch cultivatins of Tetraselmis Chui with the concentration of NaHCO3 in the range from 5 g/L to 80 g/L were conducted with the light intensity of 30000 Lux, in the Walne medium. The results indicated that the highest biomass occurred at 40 g NaHCO3 / L. 2. Effects of light intensities on the growth of Tetraselmis Chui. Batch cultivations with the light intensities of 7500, 15000 and 30000 Lux were conducted at 20 g/L of sodium bicarbonate, in the Walne medium. The results indicated that the highest biomass occurred at 15000 Lux of light. 3. Effects of initial pH of cultures on the growth of Tetraselmis Chui. Batch cultivations with the pH of 8.20, 9.39, 10.39 and 11.51 were conducted at 20 g/L of sodium bicarbonate, and 7500 Lux of light, in the Walne medium. The results indicated that Tetraselmis Chui grew quickly with pH of 8.2 and 9.39, much slowly with pH of 10.39 and stopped growing with pH of 11.51. Other factors investigated were the concentration of sodium nitrate, the concentration of NaH2PO4, the carbon sources, the light / dark period, and the composition of medium.

碩士
大葉大學
環境工程學系碩士班
97
Continuous cultivations of Tetraselmis chui were conducted in a light/dark dual-tank photobioreactor using modified Walne medium with sodium bicarbonate as the carbon source. The effects of sodium bicarbonate concentration and light intensity on growth of Tetraselmis chui were investigated in this study. Effects of sodium bicarbonate concentration in feed on growth of Tetraselmis chui were conducted with five concentrations of 0.5, 1.0, 1.5, 2.0, and 3.5 g/L. According to the results, biomass concentration increases with the increase in sodium bicarbonate concentration. Saturation for biomass concentration was occurred at higher concentration of sodium bicarbonate. However, there is no significant effect on growth rate of Tetraselmis chui with the change in sodium bicarbonate concentration. Culture pH decreases with the increase in concentration of sodium bicarbonate due to the buffer capacity of sodium bicarbonate. Effects of light intensity on growth of Tetraselmis chui were conducted with three light intensity of 4000, 4800 and 5500 lux. The results show that the higher intensity, the higher growth rate is. However, biomass concentration did not change with these three light intensities. It also observed that saturation of grow occurred at higher light intensity.

碩士
大葉大學
環境工程學系碩士班
97
Photobioreactors are normally designed to enhance the absorption efficiency of carbon dioxide and the light distribution, and to reduce self-shading in the reactor. In this study a recycle photobioreactor was designed to maintain a high concentration of microalgal biomass in the cultivation system for Tetraselmis chui by recycling culture with higher biomass concentration from the settler of the photobioreactor. The photobioreactor is consisted of a light tank and a settler. The volume of light tank is 7 L and that of settler is 12 L. Higher concentration of biomass for Tetraselmis chui was obtained in the bottom of the settler. Effects of sodium bicarbonate and recycle flow rate on growth of Tetraselmis chui were investigated. According to the result, the suitable concentration of sodium bicarbonate was 2.0 g/L for Tetraselmis chui in the recycle photobioreactor. pH of culture with higher recycle rate was lower than that with lower recycle rate. However, biomass concentration with higher recycle rate was higher than that with lower recycle rate. The better settlement occurred at the concentration of 1.5 g sodium bicarbonate and with a recycle rate of 6.96 L/hr.

碩士
國立臺灣海洋大學
生物科技研究所
94
The goal of this thesis was to expolore the arsenic methylating ability of marine and freshwater algae. Experimental analysis by hydride generation - packed coldfinger trap - atomic absorption spectrometric (HG-PCFT-AAS) showed that marine alga Tetraselmis chuii cultrued with 0.133�嵱 arsenate for 7 days generated monomethyl arsenic species (MMA) and dimethyl arsenic species (DMA), but freshwater alga Chlorella pyrenoidosa showed no methylating activity under the same condition. Tetraselmis chuii and Chlorella pyrenoidosa both showed no methylating activity when cultrued with 0.133�嵱 arsenite, a fast decrease in arsenite concentration in medium was detected after culturing Chlorella pyrenoidosa with 0.133�嵱 arsenite suggesting a rapidly binding of arsenite to algal cells. The arsenic concentration in Tetraselmis chuii medium decrease slowly indicating the weak capability of Tetraselmis chuii uptake the arsenite may be the reason why Tetraselmis chuii show no methylating activity or arsenite undergo another metabolism pathway. The addition of reducing agent GSH showed that GSH accelerate the rate of arsenic methylation and the arsenic concentration decrease more rapidly . We suggest that GSH is a important reducing factor in Tetraselmis chuii for arsenic methylation. For producing an alga species capable of methylating arsenic in freshwater, a 1011-bp cDNA carrying the complete open reading frame of mouse arsenic methyltransferase(cyt 19) was cloned into the expression vector pBIN19 under the control of a Pnos promotor and cyt19 ORF was transformed into Chlorella via infection with Agrobacterium tumefaciens. A successful transformation was confirmed by PCR,and RT-PCR showed that the expression of cyt19-m-RNA in Chlorella pyrenoidosa Analysis by HG-PCFT-AAS showed that transformed cyt19-transformed Chlorella pyrenoidosa have no arsenic methylation activity. A low but clearly detectable level of arsenic methylation was produced in cyt19-transformed with the presence of GSH and DTT. 2-D gel electrophoresis of crude extract proteins of T. chuii cultured in the presence or absence of 0.133 μM sodium arsenite for 3 days, A 40 kDa polypeptide having a PI about 6 and a 25kDa polypeptide possessing a PI about 5 were found to increase significantly in T. chuii.

Microalgae are a worldwide food source, largely consumed by humans as food or, indirectly, as feed. Their biomass might contain high levels of protein, lipids, fatty acids, amino acids, and vitamins. They are unicellular organisms capable of living in the most diverse environments, being able to grow faster than other photoautotrophs and in non-conventional growth media that do not require either potable water or arable land. With such characteristics, microalgae can become an important alternative food source in the near future since the world population is estimated to reach 10 billion people before 2050. Microalgae are produced and consumed as high-quality nutritional food, but there is an urgent need to improve existing microalgae-based products since their organoleptic properties, such as their “grassy” taste, are not consensually accepted by the consumer. The present work aims to create new strains from microalgal species already registered as novel food, namely Chlorella vulgaris and Tetraselmis chui, to improve the quality of the produced biomass and the overall consumer acceptance. For this purpose, random chemical mutagenesis using the alkylating agent ethyl methane sulphonate (EMS) was carried out to generate mutants with higher protein contents and/or lower amounts of chlorophyll. Afterwards, the best performing strains were selected using visual scoring regarding pigmentation and flow cytometry techniques. Upon implementing this selection pipeline, two C. vulgaris strains were isolated, namely the C3 strain, a non-mutagenized isolate able to grow significantly faster on solid medium compared to the wildtype (WT) culture; and a second mutant, GL3, obtained from the C3 strain. Although the C3 strain presented higher protein contents, the GL3 displayed vestigial chlorophyll contents, lower carotenoid levels, and higher protein content than the original WT strain. The GL3 strain grown under heterotrophic conditions reached higher cell concentrations as compared to the WT, strongly suggesting that the mutant strain GL3 might become a relevant source of protein, being suitable to be produced on a larger scale to generate food products with enhanced organoleptic properties.
As microalgas são organismos aquáticos unicelulares, por vezes coloniais, que normalmente são eucariotas, à exceção das cianobactérias, que são procariotas. Uma parte significativa destes organismos são fototróficos obrigatórios. embora possam ser mixotróficos, ao consumirem fontes de carbono orgânico como a glucose e a frutose, para além de fixarem carbono na forma de CO2. Podem ser ainda organismos heterotróficos facultativos, em que na falta de energia luminosa ou de carbono inorgânico, se especializaram no consumo de material orgânico. Esta forma de trofia pode ser encontrada em regiões de águas oligotróficas ou extremas, como ecossistemas cavernosos, debaixo de glaciares ou lagos subglaciais. Em termos ecológicos, a principal função desses organismos no ambiente natural é a alta carga de produção primária que são responsáveis quando fazem fotossíntese, sendo os responsáveis pela maior parte do O2 disponível no planeta, seja em ambientes aquáticos ou atmosfera. As microalgas podem variar de tamanho, sendo consideradas como tal microrganismos cuja célula está na faixa entre os 200 a 20 μm. As história evolutiva das microalgas mostra que estes organismos foram capazes de colonizar praticamente qualquer ambiente com algum tipo de habitat aquático por menor que seja. Estes ambientes vão desde grandes lagos e mares até regiões escuras e enclausuradas, incluindo ambientes com corpos d’água periódicos, mesmo que assolados por longas épocas de seca como o deserto do Atacama. A variabilidade genética e adaptabilidade das microalgas é uma questão chave para entender as caracteristicas biológicas, morfológicas e bioquímicas destes seres, que apresentam uma grande varíedade de lípidos, ácidos gordos, aminoácidos e proteínas. Essa rica composição torna os organismos deste grupo alvo de estudos para o desenvolvimento e pesquisa de produtos como ração para animais, alimento humano, produção de biodiesel, fármacos e cosméticos. Atualmente, as microalgas já são utilizadas na aquacultura como alimento vivo para criação de peixe, como biomassa para suplementação alimentar humana, fertilizantes e até mesmo como tratamento terciário de estações de tratamento de água residual de origem urbana ou industrial. A União Europeia já aceita cerca de 20 espécies diferentes de microalgas como suplemento alimentar ou alimento. Entre elas estão: Chlorella vulgaris, Arthrospira platensis (comummente designada por “Spirulina”), Dunaliella salina e Tetraselmis chui. Uma grande variedade de espécies já são atualmente produzidas comercialmente por todo o mundo, especialmente para ração animal. Porém, a cada ano que passa, o mercado para a alimentação e suplementação humana vem crescendo exponencialmente. Isso se deve ao interesse do consumirdor em uma alimentação mais saudável e natural, que diminui a procura de alimento de origem animal e aumenta a apetência por sucedâneos de origem vegetal. Aindaassim, estima-se que a produção alimentar mundial até o ano de 2050 será insuficiente, aumentando a necessidade do desenvolvimento de produtos que ocupem uma menor área para produção, sendo de alta qualidade e rendimento. A produção de microalgas se encaixa com as necessidades previstas, sendo que além de poder ser produziada em áreas inférteis e não aráveis, não necessita o uso de água potável. Tal é possível, pois há uma variada gama de espécies marinhas que podem ser domesticadas e produzidas em raceways, greenwalls e outros fotobiorreatores em que o meio de cultura pode ser feito com água do mar. A produção industrial de microalgas depende de diversas etapas, que vai desde o isolamento e identificação da espécie até produção de biomassa em larga escala. E mesmo com a tecnologia atual a produção ainda se depara com algumas limitações que tornam o processo não tão barato quanto o esperado, seja pela presença de contaminações, como pela baixa produção de algumas espécies de microalgas. Para desenvolver melhores produtos, a prospecção por diferentes espécies ou estirpes de microalgas é uma atividade constante de empresas e laboratórios especializados nos estudos e produções de microalgas. Um dos métodos para aumentar a produção é o desenvolvimento de novas estirpes, seja por domesticação e aprimoração das condições fisicoquímicas de produção ou pela mutagénese aleatória em busca de novas caracteristicas favoráveis. Algumas das propriedades mais procuradas é o aumneto da produção de proteina no conteúdo total da biomassa assim como o melhoramento das qualidades organolépticas da biomassa para aumentar ainda mais a aceitação do consumidor de produtos baseados em microalgas. Uma das causas principais para o cheiro e sabor da biomassa de microalgas ser tão acentuado é a clorofila, que, através da mutagenese, pode ser reduzida, melhorando as suas características organolépticas. Além disso, estas microalgas melhoradas tendem em apresentar uma maior percentagem de proteina total na sua biomassa, que é algo muito apreciado pela indústria alimentar. Um dos métodos para promover a mutagenese aleatória de microalgas é a aplicação de um agente alquilante como o etilmetanosulfonato (EMS) que causa uma alteração em resíduos de guanina, alterando a composição genética do material genético (e.g., pares de bases GC são mutados para AT) de forma pontual e aleatória. O facto do EMS gerar mutações pontuais torna a mutagénese mais aceitável ao consumidor do que a introdução de genes heterólogos. Além disso, perante a União Européia, tal processo não é considerado como gerador de organismos genéticamente modificados, o que facilita a sua produção à escala industrial. Essa metodologia já foi testada em diversas espécies de microalgas, como Dunaliella tertiolecta, Nanochloropsis sp. e diversas espécies de Chlorella spp. Em todos os casos, foi possível desenvolver estirpes de alta qualidade, tanto na questão de pigmentos, como lípidos, ácidos gordos e proteínas. Um dos pontos cruciais para garantir que a estirpe melhorada geneticamente continua a ser a mesma, após o processo, não sendo devido ao isolamento de algum contaminante originalmente presente na estirpe que sofreu o processo de mutagenese, é impotante realizar a identificação molecular, antes e depois de realizar a metodologia de mutagénese. A indentificação molecular se dá pela extração de material genético, seguido pela amplificação através de reação em cadeia da polimerase (PCR) de uma parte do gene 18S, que normalmente consiste em 1000 ou mais pares de base. Ainda assim, para algumas espécies de microalga a região 18S não é suficiente para se identificar diferenças interespecíficas, o que torna necessária a utilização de outros alvos como região 28S ou os espaçadores ITS1 e ITS2. Após a amplificação e sequenciação da região escolhida, é feita uma análise de similaridade com base de dados especializadas para identificar a espécie mais provável através do grau de semelhança de sequências. Para este trabalho foram utilizadas duas estirpes de espécies diferentes, Chlorella vulgaris e Tetraselmis chui, tendo estas sido cedidas pela empresa Allmicroalgae, dentro do projeto conjunto ProFuture, para a produção de estirpes melhoradas por mutagénese química aleatória através do agente EMS. Chlorella vulgaris é uma espécia já amplamente estudada desde os anos 50, e foi pioneira para a produção de biomassa para o consumo humano, já sendo perspetivada como alimento para astronautas no começo da corrida espacial. Foi prontamente aceite como "novel food" pela União Europeia, por ter um longo histórico de consumo humano durante muitos anos. A mutagénese de microalgas começou por estudos realizados em Chlorella vulgaris, para o melhor entendimento e estudo do mecanismo fotossintético, sendo provada a possibilidade de produzir estirpes de diferentes cores desde então. Tetrasselmis chui é uma das espécies de microalgas mais recentes nos termos de "novel food", e tem sido uma espécie de grande interesse por apresentar uma alta produção de biomassa e ter uma boa qualidade bioquímica, por ter uma diversidade de carotenoides, lípidos e aminoácidos de interesse nutricional. O atual projeto tem como objetivo desenvolver mutantes de Chorella vulgaris e Tetraselmis chui como uma menor taxa de clorofila e maior taxa de proteína. No caso da T. chui, no período estipulado para o projeto, não foi possível desenvolver um mutante cuja a pigmentação fosse visivelmente diferente, o que já foi explicado em outros projetos pela probabilidade de a espécie ser um organismo fotoautotrófico obrigatório, dificultando a produção de mutantes, devido ao facto de mutações no aparelho fotossintético que impliquem alterações drásticas de pigmentos serem, em geral, letais. Já no caso da Chlorella vulgaris, foi possível obter diferentes estirpes a partir da estirpe selvagem, nomeadamente a estirpe C3, que mostrou um melhor desenvolvimento em questão ao crescimento de unidades formadoras de colónias em heterotrofia e mixotrofia, mas sem revelar alterações visíveis em termos de pigmentos fotossintéticos. Assim sendo, foi selecionada a estirpe C3 para uma nova ronda de mutagénese aleatória, na qual foi possivel isolar e caracterizar a estirpe GL3 que apresentava visivelmente uma coloração amarelada. De facto, ao analisar a biomassa, foi detectado uma quantidade 20 vezes menor de clorofila e uma maior produção de proteína em gramas por 100 gramas de biomassa. A produção de uma estirpe como a GL3 foi considerada como um sucesso na aplicação da metodologia utilizada, sendo aquela enviada para o nosso parceiro empresarial para ser produzida em larga escala após uma prospecção mais detalhada da qualidade e aceitação do mercado desta biomassa inovadora.

碩士
國立臺灣海洋大學
水產養殖學系
107
This study aims to investigate the optimal growth conditions for the larval culture of Caridina brevicarpalis so that the informative results can be used as background knowledge for seed production as well as population reservation purpose. The first three experiments were conducted to investigate the effects of salinity, microalgae species and microalgae densities on the metamorphic larval survival rate and growth rate (larval stage index, LSI) in the end of each larval culture experiment. The last experiment was involved in the comparison between the two progeny sources of wild and artificially captivatedly induced spawners on their own respective larval survivals. Constant conditional setups for larval density (25 ind./L), photoperiod (12L:12D) and temperature (25±1℃) were kept all the same throughout each and every experiment described above. It was found that the highest metamorphic larval survival rate occurred at salinity level of 15‰ (53.3%) which was significantly higher than those cultivated at 20‰ (33.3%), 25‰ (19.3%), 10‰ (18.0%), and 30‰ (0%) being the lowest. The highest metamorphic larval survival rate occurred in the group of larvae received Tetraselmis. chui (62.7%) which was significantly higher than those of the rest three treatments, Thalassiosira weissflogii (4.0%) Chaetoceros muelleri (0.7%) Isochrysis galbana (0%). Of the four feeding densities of T.chui (0.5, 1.0, 2.0, 4.0) ×105 cells/ml, significant higher metamorphic larval survival rate appeared in those larvae received density either at 0.5×105cells/ml (57.3%) or 1.0×105 cells/ml (46.0%) than the other two densities at 2.0×105cells/ml (27.3%) and 4.0×105 cells/ml (0%). At salinity level of 15 ‰ and feeding density of T. chui (0.5×105 cells/ml), metamorphic larval survival rate of the artificially produced progenies (64.0%) was higher than that of the wild origin (51.3%). It is suggested that the optimum salinity of 15 ‰ and microalgal feeding densities of 0.5-1.0×105cells/ml are suitable for larval culture of C. brevicarpalis under the conditional photoperiod 12L: 12D and water temperature 25±1℃. And Larval stages of C. brevicarpalis is divided into ten stages (Zoea stage I-V, Mysis stage I-IV, Post Larva) within 24 days of culture.

碩士
國立臺灣海洋大學
生物科技研究所
93
Pentavalence arsenic and trivalence arsenic chemical compounds are toxicant in the environment and methylation of arsenic species is a detoxification pathway in some organisms. Experimental analysis by HG-GCT-AAS(Hydride Generation-Gamma Cold Trap-Atomic Absorption Spectrophotometer)showed that marine alga Tetraselmis chuii cultrued with 0.133mM arsenate for 7 days generated monomethyl arsenic species (MMA) and dimethyl arsenic species (DMA), but freshwater alga Chlorella pyrenoidosa showed no methylating activity under the same condition. For producing an alga species capable of methylating arsenic in freshwater, a 1011-bp cDNA carrying the complete open reading frame of mouse arsenic methyltransferase(cyt 19) was cloned into the expression vector pBIN19 under the control of a Pnos promotor and cyt19 ORF was transformed into Chlorella via infection with Agrobacterium tumefaciens. The cyt19 cDNA was detected by PCR in several colonies of transformed Chlorella after 2-day infection and no cyt19 cDNA could be found in all nontransformed Chlorella colonies. Whether arsenic methylating activity can be produced in transformed Chlorella awaits further investigation. Northern blot analysis of total RNA of T. chuii using cyt19 ORF as the probe detected a hybridization signal and the signal intensity increased to 142% of control after culturing T. chuii in the medium containing arsenate for 4 days and returned to normal levels 6 days later. Although three arsenate-induced polypeptides were detected by 2-D gel electrophoresis of crude extract proteins of T. chuii cultured in the presence or absence of arsenate, no induction of cyt19-like proteins was found, suggesting that the expression of arsenic methyltrnsferase in T. chuii was not regulated by arsenate.

碩士
國立臺灣海洋大學
水產養殖學系
106
The purpose of this study is to find out the most appropriate larval culture condition combinations for the long nose algae shrimp (Cardina longirostris). Stepwise of the factors pertaining to microalgae feeding density, aeration intensity, temperature, and stocking density upon the survivals and growth of larvae have been investigated through the following triplicated experiments. Condition for salinity was 20‰. Photoperiod was12L:12D. Experiment 1. Algal feeding density trial. Those fed at concentration of 3x105 cells/ml show the highest survival rate of 38.9±11.5%. which was significantly higher than that of other three treatments. There were no significant differences between 4 x105cells/ml (21.1±5.1%) and 2 x105 cells/ml (16.7 ±8.8%), and at 1 x105cells/ml (0%), the lowest. Experiment 2. Aeration intensity trial. There were no significant differences between aeration intensities of 0.7L/min (27.8±7.7%) and 0.4L/min (23.3±8.8%) on larvae survivals. But no post larvae could survive at an aeration intensity of 1.2L/min. Experiment 3. Temperature trial. Temperature at 27℃ (33.3±3.34%) was significantly higher than 29℃ (3.3±3.3%) on survivals, 25℃ (26.67±8.8%) was no significant difference among 27℃ and 29℃. Experiment 4. Larvae stocking density trial. There were no significant differences between 30 ind. /L (33.7±1.6%) and 50 ind. /L (31.1±1.6%) , Larvae stocking density of 70 ind. /L (3.5±1.5%) was significantly lower than the other two stocking densities on survivals. The study suggested that the suitable conditions for larvae cultivation at 25℃~27℃ except exceeded 29℃ with microalgae concentration of 3x105cells/ml, aeration intensity of 0.7L/min, and stocking density of 50 ind. /L enhanced the survival rate.

Dissertação de mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2017
Microalgae have been purported as a promising feedstock for the production of biodiesel, presenting a potential solution to overcome the high demand for transportation fuels. Although the production of biodiesel from microalgal biomass is feasible, the costs of production are not competitive when compared to those of fossil fuels. The biorefinery concept is a promising route to lower the costs of production of microalgal-based biofuels and enhance the final value of microalgal biomass. In this work, the potential of Tetraselmis sp. CTP4 for a biorefinery approach was evaluated, through a screening for bioactive compounds and biodiesel production. Crude (CB) and purified (PB) biodiesel were synthesized from wet microalgal biomass, and their properties were estimated based on the fatty acid methyl ester (FAME) profile obtained. Only the FAME, linoleic acid and PUFA (≥4 double bonds) contents, met the EN 14214 specifications in both. CB also met the iodine value defined in this regulation and the cetane number (CN) set in ASTM D6751 specification. Viscosity, density and cold filter plugging point (CFPP) were improved by the purification process. Concerning the biorefinery concept, three different types of activities were studied: Antioxidant, metal chelating and neuroprotection. Five different solvents were used on 2 different sources to produce the extracts: crude and spent biomass (after lipid extraction). The colloidal (CP) and water phases (WL) obtained in the crude lipid extract fractionation were also screened. The ethyl acetate, acetone and ethanol extracts from both crude and spent biomass, as well as the CP extract showed the highest antioxidant and metal chelating activities. As for the neuroprotection activity, the best results were obtained with the spent biomass extracts, using the same solvents mentioned above.
As microalgas são um grupo polifilético de organismos eucariotas unicelulares fotossintéticos. Estes organismos têm vindo a ser considerados como uma matéria prima promissora para a produção de biodiesel, apresentando, assim, uma possível solução para a crise energética que se tem observado, principalmente no que diz respeito a combustíveis líquidos para utilização em transportes motorizados. Apesar do processo de conversão da biomassa microalgal em biodiesel já ser possível, os custos associados ao mesmo não são competitivos quando comparados com combustíveis fósseis. A utilização de um conceito de biorrefinaria tem sido sugerido como uma possível solução para este problema. Este conceito depreende a exploração de todos os componentes bioquímicos da biomassa durante o processamento da mesma. O interesse comercial destes compostos pode incluir várias indústrias, como por exemplo, a indústria farmacêutica, cosmética ou alimentar. Neste trabalho foi avaliado o potencial da microalga Tetraselmis sp. CTP4 para implementação de um processo de biorrefinaria, através da exploração das propriedades do biodiesel obtido através da sua biomassa, bem como a presença de compostos bioactivos com capacidade de ação em diferentes campos. Para a obtenção do biodiesel foi realizada uma extracção lipídica, utilizando a biomassa húmida previamente cultivada, bem como uma separação do extrato obtido em três fracções distintas: a fase de hexano, posteriormente transesterificada para obter o biodiesel; a fase coloidal e a fase aquosa, sendo que as duas últimas foram utilizadas para avaliação de bioatividades. Foram depois determinadas as propriedades do biodiesel cru (CB) e purificado (PB) com base no perfil de ácidos gordos dos mesmos. Foi realizada uma comparação entre os valores obtidos para cada uma das amostras de biodiesel, e com os valores definidos nas normativas europeia (EN 14214) e americana (ATSM D6751). Apenas algumas das propriedades estudadas respeitaram, igualmente entre as duas amostras, os valores delineados nas normativas. Estas propriedades foram: a quantidade mínima de FAME (CB = 98.9%; PB = 98.7%) e a quantidade de ácido linoleico (CB = 3.73%; PB = 3.61%) presentes no biodiesel, bem como a presença de ácidos gordos polinsaturados (PUFA) com ≥ 4 ligações duplas (0% em ambos os casos). O valor de iodo do CB (114 g I/100 g) também esteve dentro dos valores definidos em EN 14214 e o número de cetano (49.3) dentro dos valores definidos em ASTM D6751. Nenhuma das amostras cumpriu com os valores definidos para a densidade (CB = 822; PB = 851 kg.m-3), e a viscosidade apenas foi cumprida dentro dos valores definidos em ASTM D6751 pelo PB (2.65 cSt). Também foi avaliado o “cold filter plugging point” (CFPP), que representa a temperatura a que o biodiesel entope filtros com poros de 0.45 μm. No entanto os valores padrão desta propriedade variam consoante o país e a estação do ano, o que torna difícil a sua comparação. De um modo geral as propriedades que foram melhoradas pela purificação do biodiesel foram a viscosidade, a densidade e o CFPP. No entanto, para propriedades como o número de cetano e o valor de iodo, observaram-se valores piores que os obtidos para CB. Como foi mencionado anteriormente, as fases coloidal e aquosa foram utilizadas para determinação das bioatividades, juntamente com extratos preparados a partir da biomassa crua e da biomassa residual, sendo que por esta última se entende a biomassa recolhida após a extração lipídica. Cinco solventes com diferentes polaridades foram utilizados no processo de extração, sendo eles o hexano, acetato de etilo, acetona, etanol e água destilada. Três tipos de atividades foram selecionados: antioxidante, quelante de metais e neuroprotectora. Várias doenças têm sido associadas com a ocorrência de stress oxidativo, nomeadamente doenças neurodegenerativas. O stress oxidativo ocorre quando se verifica um desequilíbrio entre as espécies reativas de oxigénio (ROS) e os antioxidantes presentes numa determinada célula, e dá-se a oxidação de biomoléculas constituintes da mesma. O cérebro é um órgão particularmente vulnerável a danos oxidativos, devido à escassez de antioxidantes, e altas concentrações de metais de transição (Fe3+ e Cu2+) que podem participar na formação de ROS. Apesar de existirem já vários compostos antioxidantes sintéticos em comercialização, foi recentemente demonstrado que os mesmos podem ter atividade carcinogénica. Torna-se então necessária a procura de compostos de origem natural capazes de combater este processo, sejam eles antioxidantes ou quelantes de metais. Para a atividade antioxidante determinou-se a capacidade de cada extrato para sequestrar os radicais (RSA) DPPH e ABTS. Verificou-se que os extratos com melhores RSAs foram os obtidos com acetato de etilo, acetona e etanol, tanto na biomassa crua como na residual, bem como o extrato da fase coloidal. Para as atividades quelantes de metais determinou-se a capacidade de cada extrato para quelar Fe e Cu. Os extratos que demonstraram melhor atividade foram, novamente, os descritos na atividade antioxidante. Para determinar a atividade neuroprotectora dos extratos, foi avaliada a capacidade de inibição de duas enzimas, acetilcolinesterase (AChE) e butirilcolinesterase (BChE). Estudos demonstraram que ambas as enzimas tomam parte no desenvolvimento da doença de Alzheimer (AD). Foi já aprovada a administração de inibidores destas enzimas em pacientes com Alzheimer. No entanto, estes inibidores podem provocar vários efeitos secundários bem como, em determinados casos, apresentar toxicidade, pelo que se torna importante a descoberta de novos compostos que, tendo o mesmo efeito terapêutico, reduzam ou eliminem os efeitos adversos. Os extratos que demonstraram maior capacidade inibitória foram os obtidos da biomassa residual com acetona e etanol, bem como a fase aquosa no caso da AChE e a fase coloidal no caso da BChE. Apesar dos extratos da biomassa crua terem demonstrado potencial bioactivo, para a implementação de uma biorrefinaria é necessário que os compostos bioactivos possam ser extraídos durante o processo de obtenção do biodiesel ou da biomassa residual. Uma vez que foi detetada a presença de compostos bioactivos na biomassa residual, bem como na fase coloidal recomenda-se que estes sejam fracionados, numa tentativa de obter compostos puros, dado que o facto dos extratos serem uma mistura de compostos pode alterar de alguma forma a sua atividade.

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014
Light-emitting diodes (LEDs) will become one of the world´s most important light sources and their integration in microalgal production systems (photobioreactors) needs to be considered. Microalgae need a balanced mix of wavelengths for normal growth, responding to light differently according to the pigments acquired or lost during their evolutionary history. In the present study, Nannochloropsis oculata and Tetraselmis chuii were exposed to different light qualities, and their effects on growth, biochemical components (carbohydrate, protein, total lipid and fatty acids) and morphologic traits (cell shape, size, growth phase, absorption spectrum, N-P-C elemental composition in biomass) were investigated. An additional experiment employed different LEDs in order to obtain di- and multichromatic tailored light to increase biomass production. Both N. oculata and T. chuii showed a higher maximal volumetric ash free dry weight content in the culture when exposed to blue (465 nm) and red (660 nm) light, respectively. However, balanced light quality, provided via fluorescent light (FL) and dichromatic blue and red light treatment, was found to be beneficial for biomass growth rates of both algae. Significant changes in the biochemical composition were observed among treatments. Furthermore, algae treated with monochromatic blue light (λe = 405 and 465 nm) often displayed higher nutrient uptake and different morphological traits as compared to algae exposed to red light (λe = 630 and 660 nm). It is suggested that differential response to light quality is partially influenced by observed changes in nutrient consumption and biomass productivity. In terms of biomass per input energy, the most efficient light sources were those with photon output peaks at 660 nm (e.g. LED 660 and FL for plant growth). Research and the application of LED technology to microalgal production is often hindered by inadequate light quantity measurements as well as by inadequate LED manufacture and engineering, leading to the use of inefficient LED modules, which, in turn, may affect microalgal growth and biochemistry.