Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of a reporter gene, LacZ, in the unicellular green alga Haematococcus pluvialis. By employing the micro-particle bombardment method, motile cells in the exponential phase showed transient expression of lacZ. This was detected in bombarded motile cells under the rupture-disc pressures of 3103 KPa and 4137 KPa. Transient expression of LacZ gene could not be observed in non-motile cells of this alga under the same transformation condition. No LacZ background was found in either the motile cells or the non-motile cells. The study suggests a promising potential of the SV40 promoter and the lacZ reporter gene in genetic engineering of unicellular green algae.

Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa1 : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga1

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

Intermediate pyrolysis and product identification by TGA and Py-GC/MS of green microalgae and their extracted protein and lipid components

The thermo-chemical conversion of green microalgae Chlamydomonas reinhardtii wild type (CCAP 11/32C), its cell wall deficient mutant C. reinhardtii CW15 (CCAP 11/32CW15) and Chlorella vulgaris (CCAP 211/11B) as well as their proteins and lipids was studied under conditions of intermediate pyrolysis. The microalgae were characterised for ultimate and gross chemical composition, lipid composition and extracted products were analysed by Thermogravimetric analysis (TG/DTG) and Pyrolysis-gaschromatography/mass-spectrometry (Py-GC/MS). Proteins accounted for almost 50% and lipids 16-22 % of dry weight of cells with little difference in the lipid compositions between the C. reinhardtii wild type and the cell wall mutant. During TGA analysis, each biomass exhibited three stages of decomposition, namely dehydration, devolatilization and decomposition of carbonaceous solids. Py-GC/MS analysis revealed significant protein derived compounds from all algae including toluene, phenol, 4-methylphenol, 1H-indole, 1H-indole-3methyl. Lipid pyrolysis products derived from C. reinhardtii wild type and C. reinhardtii CW15 were almost identical and reflected the close similarity of the fatty acid profiles of both strains. Major products identified were phytol and phytol derivatives formed from the terpenoid chain of chlorophyll, benzoic acid alkyl ester derivative, benzenedicarboxylic acid alkyl ester derivative and squalene. In addition, octadecanoic acid octyl ester, hexadecanoic acid methyl ester and hydrocarbons including heptadecane, 1-nonadecene and heneicosane were detected from C. vulgaris pyrolysed lipids. These results contrast sharply with the types of pyrolytic products obtained from terrestrial lignocellulosic feedstocks and reveal that intermediate pyrolysis of algal biomass generates a range of useful products with wide ranging applications including bio fuels.

Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae)

Potassium permanganate is a chemical compound widely used in aquaculture for the control and removal of parasites, and in the prevention of diseases caused by bacteria and fungi. However, this compound can be toxic to fish, being a strong oxidant. Moreover, there is no consistent information in the literature about its toxicity to non-target organisms. The purpose of this study was to evaluate the acute toxicity (LC50;96h) of potassium permanganate for tilapia, Oreochromis niloticus, and to determine its toxic effects on nontarget organisms using ecotoxicological assays performed with the microcrustacean Ceriodaphnia dubia and with the green microalgae Pseudokirchneriella subcapitata. The results showed that the concentration of 1.81 mg L-1 of potassium permanganate caused acute toxic effect in tilapia fingerlings. The ecotoxicological assays demonstrated that concentrations above 0.12 mg L-1 can cause chronic toxic effects on non-target organisms, indicating possible deleterious effects on the food chain of the aquatic ecosystem that may receive the discharge of effluents released by fish cultures treated with this chemotherapy. All toxic concentrations determined in this study were below those recommended in the literature for the use of this chemotherapy in fish cultures, demonstrating that this type of therapy should be more carefully considered in order to avoid damage to the treated fish and to the environment. (C) 2011 Elsevier B.V. All rights reserved.

Ocorrência e ecotoxicidade de desreguladores endócrinos químicos em ambientes aquáticos e em sistemas de tratamento de esgoto

A aplicabilidade de um método selecionado de medição indireta de vitelogenina (Vtg) em plasma sanguíneo de peixe, baseado na quantificação de fosfato álcali-lábil (alkali-labile phosphate-ALP) para acessar estrogenicidade em água, foi investigada na presente tese. O método foi originalmente desenvolvido para a espécie de peixe Carassius carassius (Carpa cruciana) e aplicado pela primeira vez na espécie Oreochromis niloticus (Tilápia do Nilo) no presente estudo. Com o objetivo de acessar a sensibilidade do método, em uma primeira etapa da investigação foram realizados estudos laboratoriais com soluções estoques de 17-ethinylestradiol (EE2), 17-estradiol (E2), e estrona (E1). Os efeitos destes hormônios foram investigados com base tanto na concentração quanto na carga, utilizando-se para tanto, unidades experimentais com volumes distintos (2 L e 130 L). Após a validação do método de ALP, a estrogenicidade foi avaliada nas seguintes águas contaminadas: (i) afluente e efluente de uma grande estação de tratamento de esgotos convencional (ETE) e de uma estação descentralizada de tratamento de esgoto de pequeno porte (Ecossistema Engenheirado-DEE); (ii) água superficial (SW) e água subterrânea (GW) coletadas em uma área de brejo contaminada com gasolina; (iii) água de uma lagoa urbana (LRF) da cidade do Rio de Janeiro, com alta densidade populacional e descarte clandestino de esgoto. Na segunda etapa foram analisados em microalgas os efeitos (outros que não disrupção endócrina) causados pelos hormônios EE2, E2 e E1. Os hormônios foram testados individualmente e em misturas, em culturas individuais e combinada (S+) das espécies de microalgas unicelulares P. subcapitata e D. subspicatus. Com base nos níveis de ALP para a espécie de peixe e no EC50 para as espécies de algas, os resultados mostraram que o EE2 e o E2 causaram disrupção endócrina superior e foram mais tóxicos do que o E1 para peixes e microalgas respectivamente. Quando em misturas (E+) de concentrações equivalentes (EE2:E2:E1), os estrogênios resultaram em efeito aditivo para as espécies O. niloticus e P. subcapitata, e menos que aditivo para D. subspicatus e cultivo misto de algas (S+). Culturas contendo ambas as espécies de algas (S+) por um longo período de exposição (96 h) resultaram na atenuação dos efeitos tóxicos causados pela exposição, tanto individual (EE2, E2 ou E1), quanto na mistura (E+) dos estrogênios, medidos em termos de EC50 (T0h 0,07; 0,09; 0,18; e 0,06 g mL-1; e T96h 1,29; 1,87; 5,58; e 4,61 g mL-1, respectivamente). O DEE apresentou uma maior eficiência na remoção dos disrutores endócrinos do que a ETE convencional. Foi detectada estrogenicidade em amostras da LRF, e de água SW e GW em área brejosa contaminada com gasolina. Os resultados dos ensaios sugerem que as interações (efeitos aditivos ou menos que aditivo) causadas pela mistura dos estrogênios assim como, as interações entre as espécies de algas afetaram o resultado final dos ensaios ecotoxicológicos. Um fator raramente abordado em estudos ecotoxicológicos que foi destacado na presente tese refere-se à importância de considerar não somente a concentração e a dosagem, mas também a carga aplicada e o volume das unidades experimentais. Devido à boa sensibilidade do O. niloticus quando exposto às concentrações relativamente baixas dos estrogênios, a combinação do método de ALP com os biomarcadores auxiliares (particularmente MN) pode ser um protocolo adequado para a detecção de estogenicidade e genotoxicidade respectivamente em diferentes ambiente aquáticos contaminados, como parte de um programa de monitoramento ambiental

The effect of a reduction of nitrogen and phosphorus to eutrophic and mesotrophic conditions towards silver nanoparticle (AgNP) on freshwater green algae species

We investigated the sensitivity of algae towards silver nanoparticles with OECD test medium and lower nutrient concentrations under standard test conditions to improve comparability and to exclude any other confounding factor aside nutrient levels. Two unicellular freshwater microalgae Desmodesmus subspicatus and Raphidocelis subcapitata were chosen due to their status as standard test organisms for the algae growth inhibition test and the response to changes in nutrient supply was compared. The original medium was used as the reference (standard). For the other four media, the amount of either nitrogen or phosphorus in the medium was lowered from half (50%) to one-fourth (25 %) of that of the OECD guideline, resulting in the following media: 50% N, 25% N, 50% P and 25% P medium. As test substance, the OECD reference material NM-300K was used. For this reason, the characterization of AgNP was done using DLS and Absorption spectra (UV/vis). Actual silver concentrations and ionic silver concentrations were measured at the highest test concentration used (100 µg Ag L-1) in R. subcapitata treatments only to reduce the number of samples. All tests were run according to the OECD guideline 201 with sterilized 50 mL cell culture flask. Each medium was tested using the test conditions for culturing with 3 replicates. Test concentrations for both algae species were 0, 25, 50 and 100 µg Ag L-1 for OECD, 50% P and 25% P while for both N reductions, the silver concentrations were 0, 10, 25 and 100 µg Ag L-1. Samples for determining the algal density were taken at every 24 h.

Extraction, partial characterization and susceptibility to Hg2+ of acid phosphatase from the microalgae Pseudokirchneriella subcapitata.

Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 μM Hg2+ .

Effect of two green algae species (Chlorella sp. & Scenedesmus obliquus) enriched with B group vitamins on food chain of Rutilus frisii kutum (Kamensky, 1901) fry

The Effect of two freshwater green algae species Chlorella sp. & Scenedesmus obliquus enriched (from the beginning of culture and after 96 hours) with different dosages of B group vitamins (0, 0.5, 1, and 2 ml of enriching solution per each liter of algae medium) on fecundity of Daphnia magna and growth of Rutilus frisii kutum fry were investigated in a research from spring, 2008 to autumn, 2009. First, each of the green algae species were cultured purely and massively in the Zander (Z-8+N) medium and then the nutritional value (the amount of protein, lipid, and carbohydrate) of enriched algae were meausered. In this study, enriching of Chlorella sp. & S. obliquus with a suitable mix of B group vitamins significantly improved their nutritive value. So the highest amount of nutritional value of Chlorella sp. was obtained because of enriching with dosage 0.5 ml.l-1 (366.654Kcal) and for Scenedesmus obliquus with dosage of 1 ml.l-1 (376.95Kcal). The acquired amount from control group showed an increase of respectively 42% and 11%. According to the results, increased dosages of enriching solution caused Daphnia fecundity to increase (at both stages : enrichment from the beginning of culture and after 96 hours). So the highest average of D. magna reproduction rate was obtained through being fed with Chlorella sp. and S. obliquus enriched with dosage of 2 ml enriching solution per liter of algae medium. The average fecundity of D. magna fed with Chlorella sp. enriched with dosage of 2 ml.l-1 enriching solution from the beginning of culture and after 96 hours was obtained respectively 2.128 ± 0.375 and 2.1 ± 0.69 and the average fecundity of D. magna fed with S. obliquus enriched with dosage of 2 ml enriching solution from the beginning of culture and after 96 hours was obtained respectively 2.128 ± 0.375 and 2.1 ± 0.69 which showed respectively an increase of 61 ٪, 91٪, 77 ٪, and 83٪ in proportion to the acquired amount from control group. When enriching solution was added to either algae culture medium from the beginning of culture, showed statistically significant differences (P<0.05) between dosages of 0 and 2 ml.l-1, 1 and 2 ml.l-1, and 0.5 and 2 ml enriching solution per each liter of Chlorella sp. culture medium and between dosages of 0 and 1 ml.l-1, and 0 and 2 ml enriching solution per each liter of S. obliquus culture medium. The highest average of body weight gain percentage and specific growth rate of kutum fry was obtained respectively 21.19%, 26.63%, 1.92, and 2.34 from the beginning of culture and after 96 hours with dosage of 1 ml B group vitamins per each liter of Chlorella sp. culture medium, which showed respectively an increase of 50%, 70%, 46%, and 62% in proportion to the acquired amount from control group. In the cases which Chlorella sp. were grown in the medium containing vitamin, from point of view of the average percentage of weight and specific growth rate of kutum fry significant differences were observed on the basis of the result of One-way ANOVA between dosages of 0 and 1, 1 and 2 , 0.5 and 1 ml B group vitamins per each liter. The highest average of body weight gain percentage and specific growth rate of kutum fry was obtained respectively 32.02%, 29.42%, 2.78, and 2.34 from the beginning of culture and after 96 hours with dosage of 2 ml B group vitamins per each liter of S. obliquus culture medium, which showed respectively an increase of 32%, 19%, 28%, and 17% in proportion to the acquired amount from control group. In the cases which S. obliquus were grown in the medium containing vitamin, from point of view of the average percentage of weight and specific growth rate of kutum fry significant differences were observed on the basis of the result of One-way ANOVA between dosages of 0 and 1, 0 and 2. According to the results of the present research we can say that considerable enhancement in the quality of the food of D. magna can be made by manipulation of the nutritional value of fresh water unicellular green algae with suitable mixture of B group vitamins, so that both the fecundity of D. magna will increase and the nutritional requirements of the kutum fry will be filled in this way.

Induced carotenoid accumulation in Dunaliella salina and Tetraselmis suecica by plant hormones and UV-C radiation

Carotenoids prevent different degenerative diseases and improve human health. Microalgae are commercially exploited for carotenoids, including astaxanthin and β-carotene. Two commercially important microalgae, Dunaliella salina and Tetraselmis suecica, were treated with plant hormones salicylic acid (SA) and methyl jasmonate (MJ), or by UV-C radiation (T. suecica only) and a combination thereof. Significant increases in total carotenoids were found for D. salina and T. suecica after treatment with MJ (10 μmol/L) and SA (70–250 μmol/L), respectively. T. suecica also had significant increases in total carotenoids following UV-C radiation compared to control cultures. Among the carotenoids, lutein was the highest induced carotenoid. A combination of these two treatments also showed a significant increase in total carotenoids and lutein for T. suecica, when compared to controls. Plant hormones and UV-C radiation may be useful tools for increasing carotenoid accumulation in green microalgae although the responses are species- and dose-specific and should be trialed in medium to large scale to explore commercial production.

Toxicidade e bioacumulação de cobre em micro-organismos fotoautotróficos

Microalgas e cianobactérias têm sido amplamente recomendadas para biomonitoração de metais pesados e outros poluentes, sendo considerados indicadores sensíveis às alterações ambientais e utilizados como organismos testes na regulamentação dos níveis de metal. Estes micro-organismos fotossintetizantes são produtores primários da base da cadeia alimentar aquática e são os primeiros a serem afetados pela poluição por metais pesados. O cobre é um metal normalmente considerado como nutriente essencial para a vida aquática mas pode ser tóxico para algumas espécies. Portanto, neste estudo foram avaliados o efeito tóxico e a bioacumulação de cobre (II) em quatro espécies de micro-organismos fotoautotróficos componentes do fitoplâncton dulcícola, duas cianobactérias filamentosas (Anabaena sp. e Oscillatoria sp) e duas microalgas da classe das clorofíceas (Monorraphidium sp. e Scenedesmus sp.). O meio de cultivo utilizado nos ensaios foi o ASM-1 com e sem a presença de cobre (0,6 mg/L a 12 mg Cu2+/L) onde, o efeito tóxico do metal foi monitorado por contagem celular para as microalgas e por peso seco para as cianobactérias. A bioacumulação do metal foi avaliada da mesma forma para todos os micro-organismos, através de coletas de amostras no decorrer do experimento e determinação da concentração de cobre em solução por espectrometria de absorção atômica com chama. Os resultados obtidos mostram que o efeito tóxico do metal é diretamente proporcional à concentração inicial para os micro-organismos estudados, mas que o cobre (II) foi mais tóxico para as cianobactérias que para as microalgas verdes. A bioacumulação teve uma relação direta com o efeito tóxico do metal sobre os micro-organismos. Os resultados obtidos permitem sugerir que cobre (II) tem efeito negativo no fitoplâncton, inibindo o crescimento e alterando parâmetros metabólicos como a fotossíntese. A bioacumulação do metal pode comprometer os níveis tróficos da cadeia alimentar, afetando seu transporte para seres superiores

Interações do cobre iônico com microalgas verdes

No presente trabalho foram utilizados micro-organismos fotoautotróficos para a bioacumulação de cobre em regime de batelada. Para tal, utilizou-se micro-organismos dos gêneros Ankistrodesmus, Golenkinia, Monoraphidium e Scenedesmus, em função da sua disponibilidade, facilidade de cultivo e diferenças morfológicas. Ensaios preliminares foram realizados utilizando diferentes concentrações de biossorvente, com o intuito de verificar a influência deste fator na eficiência do processo de captação. Os testes realizados em batelada incluíram também o estudo cinético e do equilíbrio, nos quais foi possível verificar que para todos os gêneros testados, o processo de captação do metal em soluções diluídas atingiu o equilíbrio quase instantaneamente. Em soluções de Cu2+ mais concentradas, observou-se diferenças entre os gêneros estudados, no tempo necessário para estabelecimento do equilíbrio. Além disso, observou-se uma alta eficiência no processo de captação de íons Cu2+ em solução por parte dos micro-organismos, com elevada captação de metal por grama de biomassa. As diferenças morfológicas entre os gêneros pareceram não influenciar significativamente a cinética e o equilíbrio do processo. Os ensaios em batelada foram realizados também empregando-se células de Golenkinia imobilizadas em alginato de cálcio, além de testes com o próprio alginato isento de micro-organismos como experimento-controle afim de verificar a influencia da imobilização nos parâmetros cinéticos e na capacidade de captação. A partir dos resultados obtidos nos ensaios em batelada com todos os biossorventes, testou-se em cada caso a aplicação de modelos cinéticos (pseudo 1 e 2 ordem) e modelos de equilíbrio (isoterma de Langmuir e Freundlich). Verificou-se que para o 4 gêneros que empregando-se células livres o modelo cujo os resultados se adequaram melhor foi o de Langmuir, enquanto que para células imobilizadas ambos modelos se mostraram adequados