Adsorption of Ni(2+), Zn(2+) and Pb(2+) onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris. I. Single metal systems

Adsorption of Ni(2+), Zn(2+) or Pb(2+) by dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris was studied as a function of contact time and initial metal concentration. The zero point of charge calculated for these biosorbents (pH(zpc) 4.0 and 3.4, respectively) and additional pH tests suggested the use of pH in the range 5.0-5.5 for the experiments. The equilibrium isotherms were evaluated in terms of maximum sorption capacity and sorption affinity. The pseudo first and second order kinetic models were considered to interpret the experimental data, and the latter best described the adsorption system. Both the Freundlich and Langmuir models were shown to well describe the sorption isotherms, thus suggesting an intermediate mono/multilayer sorption mechanism. Compared to A. platensis (q(e) = 0.354, 0.495 and 0.508 mmol g(-1) for Ni(2+), Pb(2)+ and Zn(2+), respectively), C. vulgaris behaved as a better biosorbent because of higher equilibrium sorption capacity (q(e) = 0.499, 0.634 and 0.664 mmol g(-1), respectively). The removal efficiency decreased with increasing metal concentration, pointing out a passive adsorption process involving the active sites on the surface of the biomasses. The FT-IR spectroscopy evidenced that ions removal occurred mainly by interaction between metal and carboxylate groups present on both the cell walls. (C) 2011 Elsevier B.V. All rights reserved.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: Modulation by Chlorella vulgaris prophylactic treatment

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myrelossuppression produced by the infection. Small size CFU-CM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-gamma is significantly reduced, whereas IL-10 is significantly increased after exposure to Uncontrollable stress. Treatment with CV significantly increased INF-gamma levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections. (C) 2008 Elsevier Inc. All rights reserved.

Ecotoxicity tests using the green algae chlorella vulgaris — a useful tool in hazardous effluents management

The treatment efficiency of laboratory wastewaters was evaluated and ecotoxicity tests with Chlorella vulgaris were performed on them to assess the safety of their environmental discharge. For chemical oxygen demand wastewaters, chromium (VI), mercury (II) and silver were efficiently removedby chemical treatments.Areduction of ecotoxicitywas achieved; nevertheless, an EC50 (effective concentration that causes a 50% inhibition in the algae growth) of 1.5% (v/v) indicated still high level of ecotoxicity. For chloride determination wastewaters, an efficient reduction of chromium and silver was achieved after treatment. Regarding the reduction of ecotoxicity observed, EC50 increased from 0.059% to 0.5%, only a 0.02% concentration in the aquatic environment would guarantee no effects. Wastewaters containing phenanthroline/iron (II) complex were treated by chemical oxidation. Treatmentwas satisfactory concerning chemical parameters, although an increase in ecotoxicitywas observed (EC50 reduced from 0.31% to 0.21%). The wastes from the kinetic study of persulphate and iodide reaction were treated with sodium bisulphite until colour was removed. Although they did not reveal significant ecotoxicity, only over 1% of the untreated waste produced observable effects over algae. Therefore, ecotoxicity tests could be considered a useful tool not only in laboratory effluents treatment, as shown, but also in hazardous wastewaters management.

Impact of excipients in the chronic toxicity of fluoxetine on the alga Chlorella vulgaris

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) widely used in the treatment of major depression. It has been detected in surface and wastewaters, being able to negatively affect aquatic organisms. Most of the ecotoxicity studies focused only in pharmaceuticals, though excipients can also pose a risk to non-target organisms. In this work the ecotoxicity of five medicines (three generic formulations and two brand labels) containing the same active substance (fluoxetine hydrochloride) was tested on the alga Chlorella vulgaris, in order to evaluate if excipients can influence their ecotoxicity. Effective concentrations that cause 50% of inhibition (EC50) ranging from 0.25 to 15 mg L−1 were obtained in the growth inhibition test performed for the different medicines. The corresponding values for fluoxetine concentration are 10 times lower. Higher EC50 values had been published for the same alga considering only the toxicity of fluoxetine. Therefore, this increase in toxicity may be attributed to the presence of excipients. Thus more studies on ecotoxicological effects of excipients are required in order to assess the environmental risk they may pose to aquatic organisms.

Estudo do crescimento da microalga Chlorella vulgaris numa água residual tratada, sob diferentes condições de fotoperíodo e temperatura

Dissertação apresentada à Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia, para a obtenção do grau de Mestre em Energia e Bioenergia

Viabilidade económica e energética do biodiesel produzido a partir de Chlorella vulgaris

Contemporaneamente o Homem depara-se com um dos grandes desafios que é o de efetivar a transição para um futuro sustentável. Assim, o setor da energia tem um papel fundamental neste processo de transição, com principal enfoque no setor dos automóveis, sendo este um setor que contribui com elevadas quantidades de gases de efeito estufa libertados para a atmosfera. Também a escassez dos recursos petrolíferos constitui um ponto fundamental no tema apresentado. Com a necessidade de combater esses problemas é que se tem vindo a tentar desenvolver combustíveis renováveis e neutros quanto às emissões. A primeira geração de biocombustíveis obtidos através de culturas agrícolas terrestres preenche em parte esses requisitos, porém, não atinge os valores da procura e ainda competem com a produção de alimentos. Daí o interesse na aposta de uma segunda geração de biocombustíveis produzidos de fontes que não pertencem à cadeia alimentar e são residuais mas, que mesmo assim não permitem satisfazer as necessidades de matériaprima. A terceira geração de biocombustíveis vem justamente responder a estas questões pois assenta em matérias-primas que não competem pela utilização do solo agrícola nem são usadas para fins alimentares, tendo produtividades areais substancialmente superiores às que as culturas convencionais ou biomassas residuais conseguem assegurar. A matéria prima de terceira geração são portanto as microalgas, cujas produtividades em biomassa são extremamente elevadas, para além de produtividades muito superiores em lípidos, hidratos de carbono e/ou outros produtos de valor elevado. No entanto, este tipo de produção de biocombustível ainda enfrenta alguns problemas técnicos que o tornam num processo dispendioso para competir economicamente com outros tipos de produção de biodiesel. Na linha do que foi dito anteriormente, este trabalho apresenta um estudo de viabilidade económica e energética do biodiesel produzido através da Chlorella vulgaris, apresentando as técnicas e resultados de cultivo da Chlorella vulgaris e posteriormente de produção do biodiesel através dos lípidos obtidos através da mesma. Para melhorar a colheita das microalgas, que é uma das fases mais dispendiosas, testou-se o aumento de pH e a adição de um floculante (Pax XL-10), sendo que o primeiro não permitiu obter resultados satisfatórios, enquanto o segundo permitiu obter resultados de rendimento na ordem dos 90%. Mesmo com a melhoria da etapa da colheita, o preço mínimo do biodiesel produzido a partir do óleo de Chlorella vulgaris, com as condições ótimas de cultivo e produtividades máximas encontradas na literatura, foi de 8,76 €/L, pois, na análise económica, o Pax XL-10 revelou-se extremamente caro para utilizar na floculação de microalgas para obtenção de um produto de baixo valor, como é o biodiesel. A não utilização da floculação reduz o preço do biodiesel para 7,85 €/L. O que se pode concluir deste trabalho é que face às técnicas utilizadas, a produção de biodiesel Chlorella vulgaris apenas, não é economicamente viável, pelo que para viabilizar a sustentabilidade do processo seria ainda necessário desenvolver mais esforços no sentido de otimizar a produção de biodiesel, eventualmente associando-a à produção de um outro biocombustível produzido a partir da biomassa extraída residual e/ou da recuperação de outros produtos de maior valor.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Chlorella vulgaris up-modulation of myelossupression induced by lead: The role of stromal cells

In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50 mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300 ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning. (C) 2008 Elsevier Ltd. All rights reserved.

The effects of Chlorella vulgaris in the protection of mice infected with Listeria monocytogenes. Role of natural killer cells

In this work we have demonstrated the effects of oral administration of Chlorella vulgaris (CV) on Natural Killer cells (NK) activity of mice infected with a sublethal dose of viable Listeria monocytogenes. The treatment with C. vulgaris produced a significant increase on NK cells activity in normal (non-infected) animals compared to the animals that received only vehicle (water) (p < 0.0001). Similarly, the infection alone produced a significant increase on NK cells activity, which was observed at 48 and 72 hours after the inoculation of L. monocytogenes. Moreover, when CV was administered in infected animals, there was an additional increase in NK cells activity which was significantly higher than that found in the infected groups (p < 0.0001) CV treatment (50 and 500mg/Kg) of mice infected with a dose of 3x105 bacteria/animal, which was lethal for all the non- treated controls, produced a dose-response protection which led to a 20% and 55% survival, respectively (p < 0.0001).

Metal biosorption onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris: Multi-metal systems

Binary and ternary systems of Ni2+, Zn2+, and Pb2+ were investigated at initial metal concentrations of 0.5, 1.0 and 2.0 mM as competitive adsorbates using Arthrospira platensis and Chlorella vulgaris as biosorbents. The experimental results were evaluated in terms of equilibrium sorption capacity and metal removal efficiency and fitted to the multi-component Langmuir and Freundlich isotherms. The pseudo second order model of Ho and McKay described well the adsorption kinetics, and the FT-IR spectroscopy confirmed metal binding to both biomasses. Ni2+ and Zn2+ interference on Pb2+ sorption was lower than the contrary, likely due to biosorbent preference to Pb. In general, the higher the total initial metal concentration, the lower the adsorption capacity. The results of this study demonstrated that dry biomass of C. vulgaris behaved as better biosorbent than A. platensis and suggest its use as an effective alternative sorbent for metal removal from wastewater. (C) 2012 Elsevier B.V. All rights reserved.

Pryolytic and kinetic study of Chlorella Vulgaris under isothermal and non-isothermal conditions

Algae are a new potential biomass for energy production but there is limited information on their pyrolysis and kinetics. The main aim of this thesis is to investigate the pyrolytic behaviour and kinetics of Chlorella vulgaris, a green microalga. Under pyrolysis conditions, these microalgae show their comparable capabilities to terrestrial biomass for energy and chemicals production. Also, the evidence from a preliminary pyrolysis by the intermediate pilot-scale reactor supports the applicability of these microalgae in the existing pyrolysis reactor. Thermal decomposition of Chlorella vulgaris occurs in a wide range of temperature (200-550°C) with multi-step reactions. To evaluate the kinetic parameters of their pyrolysis process, two approaches which are isothermal and non-isothermal experiments are applied in this work. New developed Pyrolysis-Mass Spectrometry (Py-MS) technique has the potential for isothermal measurements with a short run time and small sample size requirement. The equipment and procedure are assessed by the kinetic evaluation of thermal decomposition of polyethylene and lignocellulosic derived materials (cellulose, hemicellulose, and lignin). In the case of non-isothermal experiment, Thermogravimetry- Mass Spectrometry (TG-MS) technique is used in this work. Evolved gas analysis provides the information on the evolution of volatiles and these data lead to a multi-component model. Triplet kinetic values (apparent activation energy, pre-exponential factor, and apparent reaction order) from isothermal experiment are 57 (kJ/mol), 5.32 (logA, min-1), 1.21-1.45; 9 (kJ/mol), 1.75 (logA, min-1), 1.45 and 40 (kJ/mol), 3.88 (logA, min-1), 1.45- 1.15 for low, middle and high temperature region, respectively. The kinetic parameters from non-isothermal experiment are varied depending on the different fractions in algal biomass when the range of apparent activation energies are 73-207 (kJ/mol); pre-exponential factor are 5-16 (logA, min-1); and apparent reaction orders are 1.32–2.00. The kinetic procedures reported in this thesis are able to be applied to other kinds of biomass and algae for future works.

Effects of Environmental Factors on the Growth, Optical Density and Biomass of the Green Algae Chlorella Vulgaris in Outdoor Conditions

Effect of environmental factors on the growth of the Chlorella vulgaris was studied. C. vulgaris was cultivated in sterilized natural seawater enriched with F/2-Si medium. Then grow in bucket, tub and photobioreactor (PBR) in outdoor condition. The daily routine work consisted of culture checkups of optical density, biomass gains, atmosphere lux, culture lux, atmosphere temperature and culture temperature were recorded. The highest biomass yields were (3.0 μg/ml-1) in December and (2.01 μg/ml-1) in November in PBR. The highest deviation was in atmosphere lux in time 8:30 (± 117.7) and lowest deviation was in atmosphere temperature in time 15:00 (± 1.0499). Optical density (OD) indicated that the best growth of C. vulgaris in outdoor condition was obtained in 650 lux and also it increased with increasing amount of lux. Tub report of C. vulgaris showed different growing behaviors at the various concentration of light and at the different temperatures. Algal production in outdoor PBR is relatively inexpensive, but is only suitable for a few, fast-growing specie. Finally, this fact is noteworthy that in outdoor conditions, temperature and light have important role in growth of C. vulgaris in present study.

Efeitos da depleção de nitrogênio sobre a biomassa e produção lipídica lipídica de três espécies de microalgas fitoplânticas

O uso de lipídios obtidos a partir da biomassa de microalgas tem sido descrito como uma alternativa promissora para a indústria petro-diesel e envolve etapas como o cultivo de microalgas, separação da biomassa e extração de lipídios. Para viabilizar a produção em larga escala, é necessário selecionar as espécies mais produtivas, diminuir os custos de produção e determinar as condições ideais de cultivo. Os gêneros Chlorella, Desmodesmus e Ankistrodesmus apresentam características favoráveis à produção comercial, tendo sido então selecionada uma espécie de cada no presente trabalho. O objetivo do estudo foi avaliar diferentes condições de cultivo de Ankistrodesmus fusiformis, Chlorella vulgaris e Desmodesmus spinosus visando o aumento da produtividade em biomassa e lipídios totais. As algas foram identificadas e cultivadas em laboratório, em condições controladas de temperatura a 26ºC (±1), aeração por borbulhamento à pressão ambiente e luminosidade fornecida por lâmpadas fluorescentes, com intensidade de 47,25 μmol de fótons m-2.s-1 (3500 lux), fotoperiodo de 12h e pH 7, sob duas concentrações estressantes de nitrato de sódio (0,10 g/L e 0,05g/L). Os cultivos duraram em média 16 dias, sendo as curvas de crescimento construídas com dados de espectrofotometria óptica coletados a cada 48h, e a biomassa obtida ao final do cultivo por centrifugação e liofilização de cada unidade experimental. Para extração dos lipídios totais, foi utilizada a mistura de clorofórmio: metanol (1:2), segundo a metodologia de Bligh & Dyer (1959). Os tratamentos de estresse em D.spinosus resultaram em maior acúmulo lipídico, com aumento de até 149,7%, porém com drástica diminuição do crescimento e biomassa. Em C. vulgaris, nos tratamentos de estresse, verificou-se apenas ligeiro aumento do peso seco e teor de lipídios, não havendo diferença significativa entre os tratamentos e o controle. Da mesma forma, A.fusiformis não mostrou respostas significativas ao estresse pela redução de nitrato de sódio do meio, havendo ligeira diminuição do conteúdo lipídico e aumento do crescimento e biomassa. Com respostas diferentes para cada espécie estudada, evidencia-se a necessidade do conhecimento da fisiologia e autoecologia da cepa a ser cultivada em escala comercial visando à produção de ácidos graxos para fins de biodiesel.

Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plants

A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.

Produção de biomassa aquática como fonte de lípidos para biocombustíveis

Mestrado em Engenharia Química. Ramo Tecnologias de Protecção Ambiental.