Efeito das condições ambientais na produtividade lipídica da Dunaliella tertiolecta

Este trabalho teve como objectivo a optimização das condições de crescimento de biomassa algal tendo em vista a sua utilização como fonte de lípidos para biocombustíveis. Assim, procedeu-se à inoculação de duas estirpes, a Dunaliella tertiolecta (água salgada) e a Tetraselmis subcordiformis (água salobra), seleccionando-se a Dunaliella tertiolecta uma vez que esta apresentou um crescimento mais rápido. Escolhida a estirpe a usar, avaliou-se a influência da composição do meio de cultura da espécie, variando-se a concentração de macronutrientes (Magnésio, Potássio, Azoto, Fósforo) e de micronutrientes (Manganês, Zinco, Ferro, Cobalto) presentes no meio em 10 e 20 vezes, comparativamente à do meio de cultura padrão, o meio Artificial Seawater Medium with Vitamins. Avaliou-se o crescimento algal, a uma temperatura de 25 ºC ± 2 ºC, com uma intensidade de iluminação de 5000 lux (lâmpadas luz dia) e fotoperíodos 12:12 h, controlando possíveis contaminações nas culturas em estudo. Para os ensaios realizados com a Dunaliella tertiolecta, os melhores resultados para a produtividade média e máxima de biomassa, 63,06 mgbiomassa seca/L.dia e 141,79 mgbiomassa seca/L.dia, respectivamente, foram obtidos no ensaio em que se fez variar 10 vezes a concentração de azoto (sob a forma de nitrato). Os resultados mais satisfatórios para o teor lípidico e para a produtividade lipídica máxima, 33,45% e 47,43 mgóleo/L.dia respectivamente, também foram obtidos no ensaio em que se fez variar 10 vezes a concentração de azoto (sob a forma de nitrato), (com extracção dos lípidos usando o método de Bligh e Dyer). Foram testados dois solventes para a extracção de lipídos, o clorofórmio e o hexano, tendose obtido resultados superiores com o clorofórmio, comparativamente aos obtidos quando se usou hexano, com excepção do ensaio em que se aumentou 20 vezes a concentração de fósforo no meio de cultura das microalgas. Verificou-se que, em todos os ensaios foi atingido o estado estacionário sensivelmente na mesma altura, isto é, decorridos cerca de 25 dias após o início do estudo, excepto os ensaios em que se fez variar a concentração de cobalto, para os quais as culturas não se adaptaram às alterações do meio, acabando por morrer passados 15 dias. A adição dos macronutrientes e micronutrientes usados nos ensaios, nas quantidades testadas, não influenciou significativamente a produtividade lipídica, com excepção do azoto e ferro. Conclui-se que o aumento da concentração de azoto para 10x o valor padrão potencia o aumento da produtividade lipídica máxima para mais do dobro (3,6 vezes – Padrão: 13,25 mgóleo/L.dia; 10x N: 47,43 mgóleo/L.dia) e que o aumento da concentração de ferro para 10x o valor padrão potencia o aumento da produtividade lipídica máxima para aproximadamente o dobro (1,9 vezes - Padrão: 14,61 mgóleo/L.dia; 10x Fe: 28,04 mgóleo/L.dia). Nos ensaios realizados com adição de azoto ou ferro, os resultados obtidos para a concentração, teor lípidico e produtividade lipídica máxima, foram sempre superiores aos do padrão correspondente, pelo que se pode concluir que estes ensaios se apresentam como os mais promissores deste estudo, embora o ensaio mais satisfatório tenha sido aquele em que se promoveu a alteração da concentração de azoto para 10 vezes o valor padrão.

Uptake of HCO3− and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta1

Mass-spectrometric disequilibrium analysis was applied to investigate CO2 uptake and HCO3− transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO2 (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO2 and HCO3−, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO3− was the dominant inorganic C species taken up, whereas HCO3− and CO2 were used at similar rates by C. reinhardtii. The apparent affinities of HCO3− transport and CO2 uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO2 and HCO3−. For chloroplasts from C. reinhardtii, the concentrations of HCO3− and CO2 required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO3− and CO2 transporters at the chloroplast envelope membrane.

Regulation of photosynthetic pigments in micro-algae by multiple environmental factors: a dynamic balance hypothesis

Environmental effects on the concentration of photosynthetic pigments in micro-algae can be explained by dynamics of photosystem synthesis and deactivation. A model that couples photosystem losses to the relative cellular rates of energy harvesting (light absorption) and assimilation predicts optimal concentrations of light-harvesting pigments and balanced energy flow under environmental conditions that affect light availability and metabolic rates. Effects of light intensity, nutrient supply and temperature on growth rate and pigment levels were similar to general patterns observed across diverse micro-algal taxa. Results imply that dynamic behaviour associated with photophysical stress, and independent of gene regulation, might constitute one mechanism for photo-acclimation of photosynthesis.

Microalgas na Produção de Biodiesel: Extração e Composição Lipídica

The uninterrupted rise in emission of greenhouse gases open way to the use of biofuels, due to politics that focus on fuel safe, clean and renewable. The use of microalgae for biodiesel production has been described as one of the most promising sources of biomass for biofuels. The aim of this study was to evaluate the extraction and lipid profile of the microalgae Dunaliella tertiolecta, Isochrysis galbana and Tetraselsim gracilis. The extractions were performed with solvents chloroform /methanol and petroleum ether. The lipid profile was analyzed by gas chromatography after transesterification.The petroleum ether showed more efficiency in the extraction, the best result obtained was in the microalgae D. tertiolecta with 19.52% of lipid. The lipid profile analysis indicated a biodiesel stable to oxidation and elevated viscosity

Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase.

The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay

A phytotoxicity assay based on the ToxY-PAM dual-channel yield analyser has been developed and successfully incorporated into field assessments for the detection of phytotoxicants in water. As a means of further exploring the scope of the assay application and of selecting a model biomaterial to complement the instrument design, nine algal species were exposed to four chemical substances deemed of priority for water quality monitoring purposes (chlorpyrifos, copper, diuron and nonylphenol ethoxylate). Inter-species differences in sensitivity to the four toxicants varied by a factor of 1.9-100. Measurements of photosystem-II quantum yield using these nine single-celled microalgae as biomaterial corroborated previous studies which have shown that the ToxY-PAM dual-channel yield analyser is a highly sensitive method for the detection of PS-II impacting herbicides. Besides Phaeodactylum tricornutum, the previously applied biomaterial, three other species consistently performed well (Nitzschia closterium, Chlorella vulgaris and Dunaliella tertiolecta) and will be used in further test optimisation experiments. In addition to sensitivity, response time was evaluated and revealed a high degree of variation between species and toxicants. While most species displayed relatively weak and slow responses to copper, C. vulgaris demonstrated an IC10 of 51 μ g L-1, with maximum response measured within 25 minutes and inhibition being accompanied by a large decrease in fluorescence yield. The potential for this C vulgaris-based bioassay to be used for the detection of copper is discussed. There was no evidence that the standard ToxY-PAM protocol, using these unicellular algae species, could be used for the detection of chlorpyrifos or nonylphenol ethoxylate at environmentally relevant levels. © 2005 Elsevier B.V. All rights reserved.

Produção e extração da enzima anidrase carbônica e de ficobiliproteínas a partir de microalgas

Muito interesse tem sido focado no potencial biotecnológico das microalgas, principalmente devido à identificação de diversas substâncias sintetizadas por estes organismos, dentre elas a anidrase carbônica e as ficobiliproteínas. A anidrase carbônica é uma metaloenzima que catalisa a hidratação reversível do CO2 em bicarbonato com alta eficiência, sendo utilizada para captação de CO2 através de sistemas biológicos. A C-ficocianina e a aloficocianina, corantes naturais, são os dois principais componentes das ficobiliproteínas em cianobactérias e apresentam diversas aplicações dentro da indústria alimentícia, cosmética e farmacêutica. O objetivo principal desta tese foi avaliar a produção e a extração da anidrase carbônica e das ficobiliproteínas a partir de diferentes microalgas. Para isso, primeiramente foi realizado uma investigação da produção da anidrase carbônica pela microalga Dunaliella tertiolecta, onde foi estudada a extração da enzima e sua aplicação em sistemas de captura enzimática de CO2. Posteriormente foi avaliada a produção da enzima ao longo do cultivo de diferentes microalgas marinhas e dulcícolas (Dunaliella tertiolecta, Tetraselmis sueccica, Phaeodactylum tricornutum, Nannochloropsis oculata, Isochysis galbana, Chlorella vulgaris e Scenedesmus obliquus). A produção da enzima e de ficobiliproteínas, também, foi estudada para as cianobactérias Spirulina platensis LEB 52, Spirulina sp. LEB 18 e Synechococcus nidulans. Todos os cultivos foram acompanhados em termos de biomassa e pH. Por último, foi realizado um estudo de extração da enzima de P. tricornutum e extração conjunta da anidrase carbônica e de ficobiliproteínas da cianobactéria S. sp. LEB 18. Os cultivos foram realizados em frascos erlenmeyer contendo os meios Conway (marinhas), BG-11 (dulcícolas) e Zarrouk 20% (cianobactérias). Na avaliação da ruptura celular foram testadas as técnicas de maceração em gral e pistilo, agitação em vórtex com pérolas de vidro, sonicação com pérolas de vidro, homogeneizador ultrassônico, secagem, congelamento e descongelamento e a combinação de tratamentos. Maiores rendimentos de extração da enzima a partir da microalga D. tertiolecta foram obtidos utilizando tratamento ultrassônico, juntamente com baixas concentrações de biomassa úmida (0,1 e 0,2 g/L), e a mesma apresentou potencial para aplicação em processos de captação enzimática do CO2. Durante os cultivos, a microalga C. vulgaris se destacou como maior produtora da enzima anidrase carbônica, atingindo valores de atividade enzimática de 44,0 U/L. As cianobactérias apresentaram valores de atividade entre 41,6 e 45,9 U/L, sendo que a S. sp. LEB 18 foi a que apresentou maiores produções de C-ficocianina e aloficocianina no ponto de máxima atividade volumétrica, 65,9 e 82,2 µg/mL, respectivamente. A enzima extraída da biomassa de S. platensis LEB 52 catalisou a hidratação do CO2 que precipitou na forma de CaCO3. Maiores rendimentos de extração da enzima a partir das microalgas P. tricornutum e S. sp. LEB 18 foram obtidos utilizando homogeneizador ultrassônico, que foram 31,3 U/g e 25,5 U/g, respectivamente. A biomassa de S. sp. LEB 18, também apresentou potencial para a extração de ficobiliproteínas, obtendo- se altas concentrações de C-ficocianina (100,5 mg/g) e aloficocianina (69,9 mg/g). Através dos resultados obtidos, pode-se verificar a potencialidade das microalgas e das cianobactérias para produção da enzima anidrase carbônica e das ficobiliproteínas, biomoléculas de alto valor industrial. Este trabalho apresenta processos eficientes para a extração da enzima e de ficobiliproteínas tanto para escala laboratorial como industrial.

Crecimiento y ciclo de vida de la Microalga Dunaliella salina Teodoresco (CLHOROPHYTA, VOLVOCALES) de las Salinas de los Chimus (Ancash) y las Salinas de Chilca (Lima)

Para el estudio de la microalga Dunaliella salina Teodoresco, se colectaron muestras de 2 lagunas hipersalinas; las Salinas de Chimus y las Salinas de Chilca. La metodología usada fue la técnica de micropipeta, tratamiento con antibiótico y la técnica de sedimentación algal, obteniendo cultivos unialgales y axénicos. El medio usado fue medio Johnson modificado en agua de mar, utilizado concentraciones de NaCl (1 – 5M), éste permitió evaluar el crecimiento, densidad y velocidad algal a través de conteo en cámara de Newbahuer y lecturas de absorvancia en espectrofotómetro. La relación entre los métodos de conteo, se realizó con el análisis de regresión potencial. Las mejores densidades algales se observaron en cultivo de concentraciones 1M, 1,5M y 3,5M de NaCl, para las cepas de las salinas de Chimus y Chilca. La mayor densidad algal ( 4,603 x 106 cels. ml-1 equivalente al 56.4 % para la cepa de Chimus) se observó en el cultivo de 1,5M de NaCl. La velocidad de crecimiento durante la fase exponencial para ambas cepas, estuvieron entre 0,56-0,83 div. día-1, con un Td de 29-43 horas, en cámara de Newbahuer como en espectrofotómetro y a las mismas concentraciones de cultivo.

Induced β-Carotene Synthesis Driven by Triacylglycerol Deposition in the Unicellular Alga Dunaliella bardawil1

Under stress conditions such as high light intensity or nutrient starvation, cells of the unicellular alga Dunaliella bardawil overproduce β-carotene, which is accumulated in the plastids in newly formed triacylglycerol droplets. We report here that the formation of these sequestering structures and β-carotene are interdependent. When the synthesis of triacylglycerol is blocked, the overproduction of β-carotene is also inhibited. During overproduction of β-carotene no up-regulation of phytoene synthase or phytoene desaturase is observed on the transcriptional or translational level, whereas at the same time acetyl-CoA carboxylase, the key regulatory enzyme of acyl lipid biosynthesis, is increased, at least in its enzymatic activity. We conclude that under normal conditions the carotenogenic pathway is not maximally active and may be appreciably stimulated in the presence of sequestering structures, creating a plastid-localized sink for the end product of the carotenoid biosynthetic pathway.

Effects of nitrogen limitation on Dunaliella sp.–Alteromonas sp. interactions: from mutualistic to competitive relationships.

Interactions between photosynthetic and non-photosynthetic microorganisms play an essential role in natural aquatic environments and the contribution of bacteria and microalgae to the nitrogen cycle can lead to both competitive and mutualistic relationships. Nitrogen is considered to be, with phosphorus and iron, one of the main limiting nutrients for primary production in the oceans and its availability experiences large temporal and geographical variations. For these reasons, it is important to understand how competitive and mutualistic interactions between photosynthetic and heterotrophic microorganisms are impacted by nitrogen limitation. In a previous study performed in batch cultures, the addition of a selected bacterial strain of Alteromonas sp. resulted in a final biomass increase in the green alga Dunaliella sp. as a result of higher nitrogen incorporation into the algal cells. The present work focuses on testing the potential of the same microalgae–bacteria association and nitrogen interactions in chemostats limited by nitrogen. Axenic and mixed cultures were compared at two dilution rates to evaluate the impact of nitrogen limitation on interactions. The addition of bacteria resulted in increased cell size in the microalgae, as well as decreased carbon incorporation, which was exacerbated by high nitrogen limitation. Biochemical analyses for the different components including microalgae, bacteria, non-living particulate matter, and dissolved organic matter, suggested that bacteria uptake carbon from carbon-rich particulate matter released by microalgae. Dissolved organic nitrogen released by microalgae was apparently not taken up by bacteria, which casts doubt on the remineralization of dissolved organic nitrogen by Alteromonas sp. in chemostats. Dunaliella sp. obtained ammonium-nitrogen more efficiently under lower nitrogen limitation. Overall, we revealed competition between microalgae and bacteria for ammonium when this was in continuous but limited supply. Competition for mineral nitrogen increased with nitrogen limitation. From our study we suggest that competitive or mutualistic relationships between microalgae and bacteria largely depend on the ecophysiological status of the two microorganisms. The outcome of microalgae–bacteria interactions in natural and artificial ecosystems largely depends on environmental factors. Our results indicate the need to improve understanding of the interaction/s between these microbial players

Biotecnologia de Dunaliella salina para produção de B caroteno

Dissertação de mest. em Aquacultura, Unidade de Ciências e Tecnologia dos Recursos Aquáticos, Univ. do Algarve, 1997

“Effects of monospecific and mixed algal diets on survival, development and biochemical composition of Penaeus monodon larvae”

The main objective of the present work is to acquire information regarding the growth responses of P. monodon larvae (from PZ1 upto PL1) to various mono specific and mixed diets. Evaluate the nutritional quality of selected species of micro algae viz. Chaetoceros calcitrans, Dunaliella salina, Isochrysis galbana and Nannochloropsis salina, larvae at three cell concentrations 10x104 cells/ml, 25x104 cells/ml and 50x104 cells/ml. The P. monodon larvae were transported, at the Nauplius stage, to the laboratory. The larvae were stocked at density of 150 larvae per litre in 5 litre FRP tanks with 3 litres of sea water. The algal cell density given to the larvae varied. The larval stages were fed with increasing densities of algae to evaluate the relationship between the food densities, ingestion rates, development and growth of the larvae. The water quality parameters, the percentage of survival rate, the growth estimation and the algal cell count were done. Each experiment was carried out in triplicate with a control group of larvae fed with Chaetoceros calcitrans. For the estimation standard procedures were used.to P. monodon

Sustainable production of biologically active molecules of marine based origin

The marine environment offers both economic and scientific potential which are relatively untapped from a biotechnological point of view. These environments whilst harsh are ironically fragile and dependent on a harmonious life form balance. Exploitation of natural resources by exhaustive wild harvesting has obvious negative environmental consequences. From a European industry perspective marine organisms are a largely underutilised resource. This is not due to lack of interest but due to a lack of choice the industry faces for cost competitive, sustainable and environmentally conscientious product alternatives. Knowledge of the biotechnological potential of marine organisms together with the development of sustainable systems for their cultivation, processing and utilisation are essential. In 2010, the European Commission recognised this need and funded a collaborative RTD/SME project under the Framework 7-Knowledge Based Bio-Economy (KBBE) Theme 2 Programme 'Sustainable culture of marine microorganisms, algae and/or invertebrates for high value added products'. The scope of that project entitled 'Sustainable Production of Biologically Active Molecules of Marine Based Origin' (BAMMBO) is outlined. Although the Union is a global leader in many technologies, it faces increasing competition from traditional rivals and emerging economies alike and must therefore improve its innovation performance. For this reason innovation is placed at the heart of a European Horizon 2020 Strategy wherein the challenge is to connect economic performance to eco performance. This article provides a synopsis of the research activities of the BAMMBO project as they fit within the wider scope of sustainable environmentally conscientious marine resource exploitation for high-value biomolecules. © 2013 Elsevier B.V.