Das OEE-Protein 1 des Photosystem II (oxygen evolving enhancer) der Grünalge Scenedesmus obliquus besitzt Thioredoxin-Aktivität

Die Aminosäure-Sequenzierung an dem als "28 kDa-Thioredoxin f" beschriebenen Protein aus der Grünalge Scenedesmus obliquus hat gezeigt, dass dieses Protein mit dem als OEE bekannten Protein 1 aus dem Photosystem II identisch ist. Die früher postulierte Möglichkeit einer Fusion eines Thioredoxins mit einem Protein unbekannter Natur oder Insertion eines Thioredoxinfragments mit der typischen -Trp-Cys-Gly-Pro-Cys-Sequenz in ein solches Protein hat sich nicht bestätigt. Durch Anwendung einer auf das 33 kDa OEE-Protein ausgerichteten Präparationsmethode konnte gezeigt werden, dass das "28 kDa-Trx f" tatsächlich in den Thylakoidmembranen lokalisiert ist. Das Protein kann so innerhalb eines Tages in hoher Reinheit aus den Thylakoidmembranfragmenten eines Algenrohhomogenats isoliert werden; dabei bleibt die Fähigkeit des OEE-Proteins das chloroplastidäre Enzym Fructosebisphosphatase (FbPase) zu stimulieren erhalten. Mit gleichen Methoden wurden die Grünalgen Chlorella vulgaris und Chlamydomonas reinhardtii auf außergewöhnliche Proteine mit Trx-f Aktivität untersucht. Die hitze- und säurestabile Proteinfraktion aus Chlorella vulgaris enthält ein Protein mit vergleichbarer Molmasse von 26 kDa, das ähnlich wie in Scenedesmus eine Stimulation der chloroplastidären Fructosebisphosphatase zeigt. In dem hitze- und säurestabilen Proteinextrakt aus Chlamydomonas reinhardtii wird solche Aktivität nicht beobachtet. Eine Probe des rekombinanten, homogenen OEE-Proteins aus Spinat wurde auf Stimulation der chloroplastidären FbPase und NADPH-abhängigen Malatdehydrogenase (MDH) untersucht. Das Spinat OEE-Protein 1 zeigt mit diesen Enzymen keine Aktivität. Da das OEE-Protein 1 in Scenedesmus starke FbPase-Stimulation zeigt, die anderen Scenedesmus-Thioredoxine mit Molmassen von 12 kDa (Trx I und II) jedoch hohe Aktivität mit der zellulären Ribonucleotidreduktase zeigen, wird postuliert, dass das OEE-Protein die Funktion des Trx-f in vivo ersetzt.

Producción de biogás a partir de biomasa de la microalga scenedesmus sp. procedente de diferentes procesos

En este trabajo de Tesis Doctoral se ha estudiado la posibilidad de emplear las microalgas, concretamente el género Scenedesmus, como sustrato para la producción de biogás mediante digestión anaerobia, así como los residuos que se producen como consecuencia de su utilización industrial para diferentes fines. La utilización de las microalgas para la producción de biocombustibles es un tema de gran actualidad científica, en el que residen muchas expectativas para la producción a gran escala de biocombustibles que supongan una alternativa real a los combustibles fósiles. Existen numerosas investigaciones sobre la conversión a biogás de las microalgas, sin embargo aún hay poco conocimiento sobre la utilización de la digestión anaerobia como tratamiento de residuos de microalgas en un concepto de biorrefinería. Residuos que pueden ser generados tras la extracción de compuestos de alto valor añadido (p. ej. aminoácidos) o tras la generación de otro biocombustible (p. ej. biodiésel). Es en este aspecto en el que esta Tesis Doctoral destaca en cuanto a originalidad e innovación, ya que se ha centrado principalmente en tres posibilidades: - Empleo de Scenedesmus sp. como cultivo energético para la producción de biogás. - Tratamiento de residuos de Scenedesmus sp. generados tras la extracción de aminoácidos en un concepto de biorrefinería. - Tratamiento de los residuos de Scenedesmus sp. generados tras la extracción de lípidos en un concepto de biorrefinería. Los resultados obtenidos demuestran que la microalga Scenedesmus como cultivo energético para producción de biogás no es viable salvo que se empleen pretratamientos que aumenten la biodegradabilidad o se realice codigestión con otro sustrato. En este último caso, la chumbera (Opuntia maxima Mill.) ha resultado ser un sustrato idóneo para la codigestión con microalgas, aumentando la producción de biogás y metano hasta niveles superiores a 600 y 300 L kgSV-1, respectivamente. Por otro lado, el tratamiento de residuos generados tras la extracción de aminoácidos mediante digestión anaerobia es prometedor. Se obtuvieron elevados rendimientos de biogás y metano en las condiciones de operación óptimas (409 y 292 L kgSV-1, respectivamente). Aparte de la generación energética por medio el metano, que podría emplearse en la propia biorrefinería o venderse a la red eléctrica o de gas natural, reciclando el digerido y el CO2 del biogás se podría llegar a ahorrar alrededor del 30% del fertilizante mineral y el 25% del CO2 necesarios para el cultivo de nueva biomasa. Por lo tanto, la digestión anaerobia de los residuos de microalgas en un concepto de biorrefinería tiene un gran potencial y podría contribuir en gran medida al desarrollo de esta industria. Por último, una primera aproximación al tratamiento de residuos generados tras la extracción de lípidos muestra que éstos pueden ser empleados para la producción de biogás, como monosustrato, o en codigestión con glicerina, ya que son fácilmente biodegradables y el rendimiento potencial de metano puede alcanzar 218 LCH4 kgSV-1 y 262 LCH4 kg SV-1 en monodigestión o en codigestión con glicerina, respectivamente. ABSTRACT This PhD thesis explores the possibility of using microalgae, specifically the strain Scenedesmus, as substrate for biogas production through anaerobic digestion, as well as the residues generated after its use in different industrial processes. The use of microalgae for biofuels production is an emerging scientific issue. The possibility of producing biofuels from microalgae as a real alternative for fossil fuels is raising high expectations. There are several research projects on the conversion of microalgae to biogas; however, there is little knowledge about using anaerobic digestion for treating microalgae residues in a biorefinery scheme. These residues could be generated after the extraction of high value compounds (e.g. amino acids) or after the production of another biofuel (e.g. biodiesel). It is in this area in which this PhD thesis stands in terms of originality and innovation, since it has focused primarily on three possibilities: - The use of Scenedesmus sp. as an energy crop for biogas production. - Treatment of amino acid extracted Scenedesmus residues generated in a biorefinery. - Treatment of lipid extracted Scenedesmus residues generated in a biorefinery. The results obtained in this work show that the use of Scenedesmus as energy crop for biogas production is not viable. The application of pretreatments to increase biodegradability or the codigestion of Scenedesmus biomass with other substrate can improve the digestion process. In this latter case, prickly pear (Opuntia maxima Mill.) is an ideal substrate for its codigestion with microalgae, increasing biogas and methane yields up to more than 600 and 300 L kgVS-1, respectively. On the other hand, the treatment of residues generated after amino acid extraction through anaerobic digestion is promising. High biogas and methane yields were obtained (409 y 292 L kgVS-1, respectively). Besides the energy produced through methane, which could be used in the biorefinery or be sold to the power or natural gas grids, by recycling the digestate and the CO2 30% of fertilizer needs and 25% of CO2 needs could be saved to grow new microalgae biomass. Therefore, the anaerobic digestion of microalgae residues generated in biorefineries is promising and it could play an important role in the development of this industry. Finally, a first approach to the treatment of residues generated after lipid extraction showed that these residues could be used for the production of biogas, since they are highly biodegradable. The potential methane yield could reach 218 LCH4 kgVS-1 when they are monodigested, whereas the potential methane yield reached 262 LCH4 kgVS-1 when residues were codigested with residual glycerin.

Bioremediation of piggery effluents using Scenedesmus Obliquus microalga [Resumo]

Effluents from intensive pig farms present high nutrient concentration, mainly ammonium, contributing to water eutrophication and pollution. Microalgae ability to deplete inorganic nutrients makes them an efficient effluent bioremediation tool. Scenedesmus obliquus was grown in piggery effluent (without any pretreatment) diluted with tap water at 5%v/v (187±25mg/L N-NH4+) and compared with growth in synthetic Bristol media. A 21-days trial was performed in 1L bubble-column reactors illuminated by fluorescent and LED lamps(3klux). Microalgae growth was monitored through OD540nm, dry weight and Chlorophyll content and also by flow cytometry in terms of autofluorescence read in FL3 channel (>670 nm), cell size (FSC), internal complexity (SSC) and cell membrane integrity (PI). S. obliquus cells have grown slower in pig effluent (mmax=0.13-19d-1) than in Bristol media (mmax=0.46-0.50d-1) although after 15 days the biomass productivity observed for the pig waste cultivation, operated under LED (0.127gL-1d-1) was similar to those attained for the Bristol media after 8 and 12 days (0.130 and 0.129 gL-1d-1 using Fluorescent and LED lights, respectively). The Chlorophyll content was correlated to FL3 autofluorescence, with R2>0.97 for Bristol and R2>0.92 for pig waste cultures. Regarding cell size and complexity, Bristol cultures did not show significant differences along time, while cells grown on pig waste increased, attaining FSC and SSC values similar to those observed for Bristol cultures. However, pig waste led to higher percentage of cells with permeabilised membrane (up to 18%) than Bristol cultures (7%). For pig waste experiments, ammonium removal rates were 95% with final values within legal limits. S. obliquus cultivations proved to be an efficient system for direct piggery effluent bioremediation, attaining biomass productivities similar to those obtained in synthetic media. Using LED lighting enables to reduce the energy consumption while maintaining microalgae growth and bioremediation performance. Scale-up to an outdoor 150L photobioreactor is underway.

Ecological Assessment of Lentic Water Bodies of Bangalore

The restoration, conservation and management of water resources require a thorough understanding of what constitutes a healthy ecosystem. Monitoring and assessment provides the basic information on the condition of our waterbodies. The present work details the study carried out at two waterbodies, namely, the Chamarajasagar reservoir and the Madiwala Lake. The waterbodies were selected on the basis of their current use and locations. Chamarajasagar reservoir serves the purpose of supplying drinking water to Bangalore city and is located on the outskirts of the city surrounded by agricultural and forest land. On the other hand, Madiwala lake is situated in the heart of Bangalore city receiving an influx of pollutants from domestic and industrial sewage. Comparative assessment of the surface water quality of both were carried out by instituting the various physico–chemical and biological parameters. The physico-chemical analyses included temperature, transparency, pH, electrical conductivity, dissolved oxygen, alkalinity, total hardness, calcium hardness, magnesium hardness, nitrates, phosphates, sodium, potassium and COD measurements of the given waterbody. The analysis was done based on the standard methods prescribed (or recommended) by (APHA) and NEERI. The biological parameter included phytoplankton analysis. The detailed investigations of the parameters, which are well within the tolerance limits in Chamarajasagar reservoir, indicate that it is fairly unpolluted, except for the pH values, which indicate greater alkalinity. This may be attributed to the natural causes and the agricultural runoff from the catchment. On the contrary, the limnology of Madiwala lake is greatly influenced by the inflow of sewage that contributes significantly to the dissolved solids of the lake water, total hardness, alkalinity and a low DO level. Although, the two study areas differ in age, physiography, chemistry and type of inflows, they still maintain a phytoplankton distribution overwhelmingly dominated by Cyanophyceae members,specifically Microcystis aeruginosa. These blue green algae apparently enter the waterbodies from soil, which are known to harbour a rich diversity of blue green flora with several species common to limnoplankton, a feature reported to be unique to the south Indian lakes.Chamarajasagar water samples revealed five classes of phytoplankton, of which Cyanophyceae (92.15 percent) that dominated other algal forms comprised of one single species of Microcystis aeruginosa. The next major class of algae was Chlorophyceae (3.752 percent) followed by Dinophyceae (3.51 percent), Bacillariophyceae (0.47 percent) and a sparsely available and unidentified class (0.12 percent).Madiwala Lake phytoplankton, in addition to Cyanophyceae (26.20 percent), revealed a high density of Chlorophyceae members (73.44 percent) dominated by Scenedesmus sp.,Pediastrum sp., and Euglena sp.,which are considered to be indicators of organic pollution. The domestic and industrial sewage, which finds its way into the lake, is a factor causing organic pollution. As compared to the other classes, Euglenophyceae and Bacillariophyceae members were the lowest in number. Thus, the analysis of various parameters indicates that Chamarajasagar reservoir is relatively unpolluted except for the high percentage of Microcystis aeruginosa, and a slightly alkaline nature of water. Madiwala lake samples revealed eutrophication and high levels of pollution, which is clarified by the physico–chemical analysis, whose values are way above the tolerance limits. Also, the phytoplankton analysis in Madiwala lake reveals the dominance of Chlorophyceae members, which indicate organic pollution (sewage being the causative factor).

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

Microalgal community structure in experimental carp-pangasiid catfish polyculture ponds

Microalgal community structure in experimental carp-pangasiid catfish polyculture ponds under four different stocking rates (treatments) each with three replications in the Field Laboratory of the Faculty Fisheries, Bangladesh Agricultural University, Mymensingh was studied. A total of 38 microalgal genera were identified under four major groups: 18 genera belong to Chlorophyceae, 9 to Cyanophyceae, 8 to Bacillariophyceae and 3 to Euglenophyceae. Chlorophyceae was abundant in all treatments followed by Cyanophyceae, Bacillariophyceae and Euglenophyceae throughout the study period. The cell densities of total microalgal population varied between 51.66x10^3 cells/L in June in T1 and 126.4x10^3 cells/L in August in T2. The appearance of Microcysris, Oscillatoria, Gomphospheria, Hildenbrandia, Chlorella, Scenedesmus, Cyclotella, Navicula, Nitzschia, Euglena and Phacus as dominant genera throughout the study period may related to sufficient nutrient availability, good light conditions and high growth rate of these genera. Water quality parameters of the experimental ponds were within suitable range for microalgal production and fish culture though the nutrient (nitrate-nitrogen and phosphate-phosphorus) concentrations were high. The factors involved in structuring a phytoplankton community arise from the relationship generated by physical, chemical and biological conditions especially the stocked planktivorous carps. Microalgal bloom formation is very common in pangasiid catfish monoculture ponds but in the present study bloom was not formed and the algal species diversity was found to be slightly increased with the study period. The introduction carps of carps in the experimental ponds might have helped in controlling the microalgal bloom formation and maintenance of the species diversity.

The comparison of inhibitory effects of barley and rice straws to control of algae and aquatic weeds in fish ponds

Bioassays were carried out to consider the relative effect of barley and rice straw extracts on the growth of 12 problematic freshwater algae. The laboratory results demonstrated that growth of Microcystis aeruginosa; Aphanizomenon flos–aquae, Anabaena flos–aquae, Synura petersenii, and Dinobryon sertularia were all inhibited, whereas Oscillatoria tenuis, Asterionella formosa, Spirogyra sp., and Hydrodictyon reticulatum grown were stimulated by the barley straw extract. No significant effect was also found on the growth of Chlorella kessleri, Scenedesmus subspicatus, and Navicula pelliculosa.

斜生栅藻中虾青素的积累过程及其光合活性变化

分析了斜生栅藻(Scenedesmus obliquus)在光温(30℃,180μmol/m2·s)胁迫条件下积累虾青素的过程,观察了该过程中细胞形态及细胞光合生理的变化。胁迫条件下,细胞在48h内生成并积累了包括海胆酮、角黄素、金盏花黄素和金盏花红素在内的多种次生类胡萝卜素,并合成了虾青素及其酯。该过程中,细胞形态由两端尖细变得不规则、膨大,原来由4、8个细胞组成的定形群体变为游离的单个细胞或2个细胞组成的群体。藻细胞光合速率在24h内先下降后上升,而后又呈现下降趋势,从34.29μmolO2/mg C

人工湿地-池塘复合养殖系统中浮游藻类生态特征

采用放养了斑点叉尾鮰苗种和少量白鲢的人工湿地-池塘复合养殖系统研究各养殖塘浮游藻类的生态特征,并通过对不同密度养殖塘的比较探讨人工湿地对鱼塘中浮游藻类生态结构的调节作用。结果表明,塘中共鉴定出浮游藻类7门63属142种,其中绿藻门34属60种,硅藻和蓝藻分别有10属34种、10属30种,裸、隐、甲、金藻门种类较少,共9属18种。各养殖塘优势种类主要为四尾栅藻(Scenedesmus quadricauda)、衣藻(Chlamydomonas sp.)、二角盘星藻(Pediastrum duplex)、小环

武汉东湖湖水的藻类生长潜力(AGP)测试

在东湖湖水样品中添加排入东湖的主要污水或营养物(氮和磷)进行藻类测试,观察它们对斜生橱藻(Scenedesmus obliquus)的生长促进作用.生长反应与添加的污水浓度成正比,其SC_(20)(促进20％增长的浓度)为0.5—4％.单独添加氮或磷,在高浓度情况下也很少促进藻类生长,但共同添加时大多有促进作用.东湖为一严重富营养型湖泊,为了控制其富营养化进程,污水截流应是首先要采取的一项措施.

利用水生植物清除水体中放射性物质的研究 Ⅱ.斜生栅藻对放射性物质的吸收和去除

以斜生栅藻[Scenedesmus obliquus (Turp.)Kutz.]为材料,进行了藻类对三种放射性物质(K2HPO4-P32,NaI-I131,SrCl-Sr89)的吸收与去除试验。试验结果表明,斜生栅藻对这些放射性物质的吸收能力强,速度快,具有较好的去除效果。用超声波、红外线和γ射线对藻类进行预处理,可以在不同的程度上提高吸收能力和去除效果。

Grazing on toxic and non-toxic Microcystis aeruginosa PCC7820 by Unio douglasiae and Corbicula fluminea

To explore the potential grazing effects of mussels on Microcystis aeruginosa, a common bloom-forming phytoplankton, Unio douglasiae and Corbicula fluminea were fed with Scenedesmus obliquus, toxic and non-toxic strains of Microcystis aeruginosa as single food and as mixtures in the laboratory. When fed with single foods, U. douglasiae has similar clearance rates on the three algae populations, while C. fluminea has significantly lower clearance rate on toxic M. aeruginosa than those on the other two algae populations. When fed with mixture foods, both the mussels show significantly higher clearance rates than on single foods. The clearance rates of U. douglasiae on the different food mixtures are not significantly different, and C. fluminea has a significantly lower clearance rate on the toxic food mixtures than that on non-toxic food mixtures. Although the relative lower clearance rates of C. fluminea on toxic food, we may still deduce that both the mussels can exert grazing pressure on phytoplankton. The deduction is supported by the composition of the excretion products. The excretion products (faeces and pseudofaeces) of both mussels contained mainly S. obliquus. In both mixed-food treatments, the ratios of S. obliquus to M. aeruginosa in the excrete products are significantly higher than those in the foods. Therefore, it can be concluded that both mussels prefer M. aeruginosa to S. obliquus, and can cause grazing pressure on M. aeruginosa.

Development of tolerance against toxic Microcystis aeruginosa in three cladocerans and the ecological implications

This is the first experimental study to compare difference in the development of tolerance against toxic Microcystis among multi-species of cladocerans (Daphnia, Moina and Ceriodaphnia) pre-exposed to two M. aeruginosa PCC7820 strains (MC-containing and MC-free). Zooplankton were divided into S population (fed Scenedesmus), M-F population (fed Scenedesmus + MC-free Microcystis), and M-C population (fed Scenedesmus + MC-containing Microcystis). M-F and M-C populations were pre-exposed to Microcystis strains for 4 weeks, and their newborns were collected for experiments. A pre-exposure to MC-containing or MC-free Microcystis increased tolerance against toxic Microcystis. The marked increases in survival rate and median lethal time (LT50, 100-194% increase) in the M-C population of Ceriodaphnia suggest that small-sized cladocerans may develop stronger tolerance against Microcystis than large-sized ones when both groups are exposed to toxic Microcystis. This may explain why dominant Daphnia is usually replaced by small-sized cladocerans when cyanobacteria bloomed in summer in eutrophic lakes. (c) 2005 Elsevier Ltd. All rights reserved.