Produção de bioetanol a partir da microalga Scenedesmus obliquus

Dissertação para obtenção do Grau de Mestre em Energia e Bioenergia

Rheological behavior of Chlorella sp. e Scenedesmus sp. cultures in different biomass concentrations

Studies involving the use of microalgae are increasingly intensifying for the potential they present to produce biofuels, because they are a renewable energy source that does not compete directly with food production, and because they enable the obtaining of a fuel with less environmental impact when compared to fossil fuel. In this context, the use of microalgae is directly associated to its capacity to be produced on a large scale and to be extracted from the culture medium. Rheological studies are important for obtaining the information needed in the elaboration of projects and equipment that will be used in various operations existing in systems of production and extraction of algal biomass. In the evaluation of different levels of dry biomass concentration, studies have been conducted of the rheological behavior of cultures of Chlorella sp. BR001 and Scenedesmus sp. BR003. The Power Law model adjusted well to the data of shear stress as a function of strain rate. In all concentrations the cultures showed non-Newtonian behavior. It was observed to Scenedesmus sp. BR003 little effect of biomass concentration on the apparent viscosity and shear stress.

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.

Effects of temperature, sestonic algae features, and seston mineral content on cladocerans of a tropical lake

The effects of temperature on the life table, and of seston quality on the individual growth and reproduction of cladocerans from a tropical lake were tested in the laboratory. Life-table experiments were carried out at 17 degrees C, 23 degrees C, and 27 degrees C. Growth bioassays tested the influence of natural seston fractions, separated by net filtration, on cladocerans. The treatments were: (1) total seston plus Scenedesmus spinosus (1 mg C.L(-1)), (2) seston <= 36 mu m, and (3) seston >36 mu m. Phytoplankton composition, density, and biomass were evaluated during growth experiments, together with sestonic carbon, nitrogen, and phosphorus concentrations. The intrinsic rates of natural increase were higher for Moina micrura and Daphnia ambigua at 27 degrees C compared to 17 degrees C. The age at first reproduction of both species was delayed at 17 degrees C. Growth rates and fecundity of M. micrura were higher in the seston fraction <= 36 mu m than in the fraction > 36 mu m. Higher growth rates and fecundity of Moina minuta were observed in the seston enriched with the green alga in comparison to the seston <= 36 mu m and > 36 mu m. Bosmina longirostris was unable to reproduce at 17 degrees C and to grow in the seston > 36 mu m in one experiment. High densities and/or biomass of large colonial and filamentous algae present in the larger seston fraction could have contributed to reduce growth and reproduction. Episodes of food-quantity limitation may occur, but there was no evidence of mineral limitation, although seston C:P and C:N ratios were always above the limiting values assumed for temperate water bodies. The C:P and C:N ratios arc highly influenced by carbon that originates primarily from resuspended detritus from the lake.

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5-76.0% and 13.2-19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m(biogas)(3)/kgCOD(i)) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO(2) was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 +/- 0.006 g(DM)/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%. (C) 2009 Elsevier Ltd. All rights reserved.

Produção de biohidrogénio a partir de diferentes fontes de carbono, por fermentação anaeróbia

Dissertação para a obtenção do Grau de Mestre em Energia e Bioenergia

Determination of microalgae cellular composition after phycoremediation of swine wastewaters.

Phycoremediation of swine wastewaters has been widely reported as an attractive tertiary treatment system, that effectively removes the excessive nutrient loadswhilst offering a valuable source of feedstock biomass. Digestate from an upflow anaerobic sludge blanket (UASB, 6%v/v) and a nitrification reactor (NR; 50% v/v) were used as culturing media to microalgae. Experiments were carried out in lab scale photobioreactors (PBRs) using a consortia of Chlorella and Scenedesmus. Ammonia (44 to 90%) and phosphorus (77%) were efficiently removed from both effluents tested after 4 days. Microalgae biomass harvested from the UASB effluent showed 57, 34 and 1% of proteins, carbohydrates and lipids, respectively. Comparatively, the cellular composition of microalgae grown on NR effluent had lower protein (43%) but higher carbohydrate (42%) contents. Negligible difference in lipid fraction was observed independently of the effluents tested. The results suggest that the biomass harvested from phycoremediation of swine wastewaters can offer a valuable protein and carbohydrate feedstock for nutritional and biotechnological applications.

FOOD CONCENTRATION AND TEMPERATURE EFFECTS ON LIFE CYCLE CHARACTERISTICS OF TROPICAL CLADOCERA (Daphnia gessneri Herbst, Diaphanosoma sarsi Richard, Moina reticulata (Daday)): I. DEVELOPMENT TIME

The effects of food concentration and temperature on embryonic and postem-bryonic duration of three tropical species, Daphnia gessneri(1.5mm), Diaphanosoma sarsi(1.2mm) and Moina reticulata(0.8mm), were investigated as part of life cycle studies which included growth, body size and reproduction. These are the very first experimental studies undertaken on these species. The long-term growth experiments were performed under controlled laboratory conditions at all combinations of temperature (22"C, 27"C and 32"C) and constant food concentration (0.03, 0.05, 0.10, 0.25, 0.50 and 1.00 mgC/L) of the unicellular green alga Scenedesmus acutus.Animals were examined twice daily throughout their life cycle from the neonate to third adult instar. In all three species, temperature exerted the most powerful influence on embryonic duration but there was also a smaller food effect. In D. gessneri,postembry-onic durations remained more or less the same at food levels 0.25 mgC/L but were influenced by temperature. At food concentrations of 0.1 mgC/L or lower, postembryonic durations became increasingly prolonged, particularly at high temperatures. This threshold concentration is affected by temperature: in D. gessneri,it was 0.1 mgC/L at 22oC and 27oC but higher at 32oC (between 0.25 and 0.50 mgC/L). At the same temperature of 27oC, the food threshold level varied between species: it was higher (0.25 mgC/L) for D. sarsiand lower (0.05 mgC/L) for M. reticulatacompared with D. gessneri(0.1 mgC/L). In both embryonic and postembryonic durations there is a body size effect as the absolute durations were longest in the largest species and shortest in the smallest species In all three species, prolongation of postembryonic duration at combinations of high temperature and lowered food levels was accompanied by increased number of juvenile instars.

Qualidade nutricional de três espécies de clorofícias cultivadas em laboratório

Três espécies de algas clorofíceas, Scenedesmus quadricauda (Brèbisson, 1835), Ankistrodesmus gracilis (Korsikov, 1953) e Pediastrum duplex (Meyen, 1829), foram cultivadas no laboratório, usando como meio de cultura adubo químico N:P:K na proporção 20:5:20, respectivamente. O valor nutricional das algas foi avaliado pela concentração de clorofila-a e pelo carbono orgânico. As três espécies algais apresentaram alto valor nutricional, particularmente Scenedesmus quadricauda, que apresentou a maior percentagem de carbono em relação ao peso seco (21%), sendo considerada como alimento adequado para os organismos filtradores.

Plancto e hidrobiologia sanitária de tanques tropicais com dáfnias e rotíferos

The engineers of the modern University City are constructing a graceful bridge, named PONTE OSWALDO CRUZ, that crosses a portion of the Guanabara Bay (Fig. 1). The work at west pillar stopped for 3 years (The concret structure in Est. 1). As it will be seen from n.º 1 — 5 of the fig. 1, Est. I, the base of the structure will have five underground boxes of reinforcement, but, to-day they are just like as five uncovered water ponds, until at present: May 1963. (Est. I — fig. 3, n.º 3 — pond n.º 3; A. — old level of the water; B.— actual level of the water; c.— green water; E.— mass of bloom of blue algae Microcystis aeruginosa). Soon after SW portion, as 5 cells in series, of the pillar abutments, and also the NE portion nearly opposite in the Tibau Mount will be filled up with earth, a new way will link Rio City and the University City. We see to day Est. I, fig. 1 — the grasses on the half arenous beach of the Tibau Point. These natural Cyperaceae and Gramineae will be desappear because of so a new road, now under construction, when completed will be 33 feet above the mean sea level, as high as the pillar, covering exactly as that place. Although rainfall was the chief source of water for these ponds, the first water (before meterorological precipitations of whatever first rain it might fall) was a common tap water mixed with Portland Cement, which exuded gradually through the pores of the concret during its hardenning process. Some data of its first cement water composition are on the chemical table, and in Tab. n.º 4 and "Resultado n.º 1". The rain — receiving surface of each pond were about 15 by 16 feet, that is, 240 square feet; when they were full of water, their depth was of 2 feet 3", having each pond about 4,000 gallons. Climatic conditions are obviously similar of those of the Rio de Janeiro City: records of temperature, of precipitation and evaporation are seen on the graphics, figs. 2, 3, 4. Our conceptions of 4 phases is merely to satisfy an easy explanation thus the first phase that of exudation of concrete. We consider the 2nd. phase formation of bacterian and cyanophycean thin pellicel. 3rd. phase - dilution by rains, and fertilisation by birds; the 4th phase - plankton flora and fauna established. The biological material arrived with the air, the rains, and also with contaminations by dusts; with big portion of sand, of earth, and leaves of trees resulted of the SW wind actions in the storming days (See - Est. I, fig. 3, G. - the mangrove trees of the Pinheiro Island). Many birds set down and rest upon the pillar structure, its faeces which are good fertilizers fall into the ponds. Some birds were commonly pigeons, black ravens, swallows, sparrows and other sea mews, moor hens, and a few sea birds of comparatively rare occurence. We get only some examples of tropical dust contaminated helioplankton, of which incipient observations were been done sparcely. See the systematic list of the species of plankters. Phytoplankters - Cyanophyta algae as a basic part for food of zooplankters, represented chiefly by rotiferse, water-fleas Moinodaphnia and other Crustacea: Ostracoda Copepoda and Insecta: Chironomidae and Culicidae larvae. The polysaprobic of septic irruptions have not been done only by heating in summer, and, a good reason of that, for example: when the fifth pond was in polysaprobic phase as the same time an alike septic phase do not happened into the 3rd. pond, therefore, both were in the same conditions of temperature, but with unlike contaminations. Among the most important aquatic organisms used as indicatiors of pollution - and microorganisms of real importance in the field of sanitary science, by authorities of renown, for instance: PALMER, PRESCOTT, INGRAM, LIEBMANN, we choose following microalgae: a) The cosmopolite algae Scenedesmus quadricuada, a common indicator in mesosaprobio waters, which lives between pH 7,0 and it is assimilative of NO[3 subscripted] and NH[4 subscripted]. b) Species of the genus Chlamydomonas; it is even possible that all the species of theses genus inhabit strong-mesosaprobic to polysaprobic waters when in massive blooms. c) Several species of Euglenaceae in fast growing number, at the same time of the protozoa Amoebidae, Vorticellidae and simultaneous with deposition of the decaying cells of the blue algae Anacystis cyanea (= Microcystis) when the consumed oxygen by organic matter resulted in 40 mg. L. But, we found, among various Euglenacea the cosmopolite species (Euglena viridis, a well known polysaprobic indicatior of which presence occur in septic zone. d) Analcystis cyanea (= M. aeruginosa) as we observed was in blooms increasing to the order of billions of cells per litter, its maximum in the summer. Temperatures 73ºF to 82ºF but even 90ºF, the pH higher than 8. When these blue algae was joined to the rotifer Brachionus calyflorus the waters gets a milky appearance, but greenished one. In fact, that cosmopolite algae is used as a mesosaprobic indicator. Into the water of the ponds its predominance finished when the septic polysaprobic conditions began. e) Ankistrodesmus falcatus was present in the 5th pond from 26the. April untill the 26th July, and when N.NH[4 subscripted] gets 1.28 mg. L. and when chlorinity stayed from 0.034 to 0.061 mg. L. It never was found at N.NH[4 subscripted] higher than 1 mg. L. The green algae A. falcatus, an indicatior of pollution, lives in moderate mesosaprobic waters. f) As everyone knows, the rotifer eggs may be widely dispersed by wind. The rotifer Asplanchna brightwelli in our observation seemed like a green colored bag, overcharged by green cells and detritus, specially into its spacious stomach, which ends blindly (the intestine, cloaca, being absent). The stock of Asplanchna in the ponds, during the construction of the bridge "PONTE OSWALDO CRUZ" inhabits alkaline waters, pH 8,0 a 8,3, and when we observed we noted its dissolved oxygen from 3.5 to 4 mg. L. In these ponds Asplanchna lived in 0,2 P.PO[4 subscripted]. (Remember the hydobiological observations foreign to braslian waters refer only from 0.06 to 0,010 mg. L. P.PO[4 subscripted]; and they refer resistance to 0.8 N.NH[4 subscripted]). By our data, that rotiger resist commonly to 1.2 until 1.8 mg. L.N.NH[4 subscripted]; here in our ponds and, when NO[2 subscripted] appears Asplanchna desappears. It may be that Asplanchna were devoured by nitrite resistant animals of by Culicidae or other mosquitoes devoured by Due to these facts the number and the distribution of Asplanchna varies considerabley; see - plates of plankton successions. g) Brachionus one of the commonest members of class Rotatoria was frquently found in abundance into the ponds, and we notice an important biological change produce by the rotifer Brachonus colyciflorus: the occurence of its Brachionus clayciflorus forms pallas, is rare in Brazil, as we know about this. h) When we found the water flea MOinodaphnia we do not record simultanous presence of the blue algae Agmenellun (= Merismopedia).