A necessidade de se encontrar um substituto ao combustível fóssil tem impulsionado o desenvolvimento de novas fontes de biomassa para os biocombustíveis. Atualmente uma fonte de biomassa alternativa que vêm ganhando destaque são as microalgas, micro-organismos fotossintéticos capazes de capturar o CO2 atmosférico e acumular altos teores de lipídeos em sua biomassa, tornando-os muito atrativos como fonte de matéria-prima para a síntese de biodiesel. Deste modo, o presente trabalho teve como objetivo estudar os efeitos dos fatores NaNO3, CO2, intensidade luminosa e profundidade no cultivo da microalga marinha Chlorella sp visando o acúmulo de lipídeos dessa microalga em sua biomassa para a produção de biodiesel. O trabalho foi dividido em três etapas principais: na primeira foram realizados os cultivos nos quais os fatores de interesse foram avaliados em dois níveis de operação, NaNO3 (0,25 e 0,75 g/l), CO2 (5 e 10 %), intensidade luminosa (0,85 e 14,5 klux) e profundidade (5 e 10 cm); na segunda etapa foram realizadas as extrações lipídicas da biomassa para a quantificação dos teores lipídicos; e na terceira etapa realizou-se a síntese do biodiesel por via química a partir dos lipídeos extraídos da biomassa. Os experimentos que compreenderam a etapa dos cultivos foram realizados segundo um planejamento de Taguchi L8, considerando como variáveis respostas concentração de biomassa e teor lipídico. O melhor ajuste para maximizar a concentração de biomassa e teor lipídico foi de 0,85klux de intensidade luminosa, 5% de CO2, 10 cm de profundidade e 0,42g.L-1 de NaNO3 que forneceu uma concentração de biomassa de 2,04 g.L-1 com 15,04% de teor lipídico. O perfil lipídico apresentou uma proporção de 60 % entre ácidos graxos saturados, 31,7% entre ácidos monoinsaturados e 8,26 % de ácidos poli-insaturados e a reação de síntese do biodiesel atingiu uma conversão de 78,4%. / The necessity of finding a substitute to fossil fuel has enhanced the development of new biomass sources for biofuels. Currently, an alternative source of biomass that has acquiring prominence are microalgae, which are photosyntetical microorganisms able to capture the atmospheric CO2 and accumulate high content of lipids in their biomass, making them a very attractive feedstock source for biodiesel synthesis. In such a way, the present work had the objective of studying the effect of the following factors: NaNO3, CO2, luminous intensity and depth in the cultivation of marine microalga Chlorella sp, aiming the lipid accumulation of this microalga in its biomass for biodiesel production. The project was divided in three main steps: in the first one, the experiments were performed to evaluate the following factors in two levels of operation, NaNO3 (0,25 and 0,75 g/l), CO2 (5 and 10 %), luminous intensity (0,85 and 14,5 klux) and depth (5 and 10 cm); in the second step, the lipid extractions from the biomass were performed to quantify the lipid contents; and in the third one, the synthesis of biodiesel via chemical route was performed utilizing the lipids extracted from the biomass. The experiments regarding the cultivation steps were performed according to a Taguchi L8 experimental design, considering the biomass concentration and lipid content as response variables. The best fit for maximization of biomass concentration and lipid content was 0,85 klux of luminous intensity, 5% of CO2, 10 cm of depth and 0,42g/l of NaNO3, which provided a biomass concentration of 2,04 g/l with 15,04% of lipid content. The lipid profile showed a proportion of 60% of saturated fatty acids, 31,7% of monounsaturated fatty acids and 8,26% of polyunsaturated fatty acids and the reaction of biodiesel synthesis yielded a conversed of 78,4%.

Orientador: Antonio Carlos Monteiro / Banca: Ernani Pinto Júnior / Banca: Eliana Gertrudes Macedo Lemos / Banca: Ana Teresa Lombardi / Banca: Jackson Antonio Marcondes de Souza / Resumo: A microalga Haematococcus pluvialis é conhecida como o microrganismo que apresenta maior rendimento final de astaxantina em sua biomassa, quando submetida a fatores de estresse celular eficientes. Contudo ainda apresenta uma característica de crescimento desfavorável quando comparada a outras espécies de microalgas cultivadas em escala comercial, visto que possui uma taxa de crescimento intrínseca, resultando em baixa densidade celular ao final dos cultivos. Assim, o rendimento final do pigmento é relativamente baixo. Neste sentido, este estudo teve por objetivo avaliar a resposta de Haematococcus pluvialis ao processo de indução à carotenogênese por meio de estresse luminoso e nutricional, e promover o aumento de biomassa vegetativa da microalga através da adição da rizobactéria promotora de crescimento em plantas, Azospirillum brasilense V6, estirpe produtora do fito hormônio AIA (ácido indol - acético), em cultivos estanques, visando obter maior rendimento final de astaxantina. O trabalho foi dividido em duas fases. Primeiramente, no cultivo referente ao controle (contendo apenas H. pluvialis) foram aplicadas duas combinações de fatores de estresse celular para indução a síntese de astaxantina pela microalga, visando a escolha do método de estresse a ser aplicado na fase seguinte. As células de H. pluvialis foram submetidas ao estresse por alta intensidade luminosa (350 μmol photons m-2 s-1) ou ao mesmo estresse luminoso aliado a suplementação com CO2 (4%) na aeração fornecida na fase exponencial de crescimento. Avaliou-se, entre outros fatores de resposta celular, a concentração final de astaxantina na biomassa após 10 dias de estresse. As concentrações do pigmento foram 4,15 e 17,66 mg g-1, respectivamente, para as culturas submetidas ao estresse luminoso e para... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The microalgae Haematococcus pluvialis is known as the microorganism that has a highest final yield of astaxanthin in its biomass, when induced by efficient stress factors. However, still presents a characteristic of unfavorable growth when compared to other species of microalgae grown on a commercial scale, once it has an intrinsic growth rate that provides low cell density in the culture. Thus, the final yield of the pigment is relatively low. Thus, this study aimed to evaluate the response of Haematococcus pluvialis submitted to carotenogenesis induction process through light and nutrient stress, and promotion of vegetative microalgae biomass increase through the addition of the growth promoter bacteria in plants, Azospirillum brasilense V6, a strain that produce the phyto hormone IAA (indole - acetic acid). The experiments were performed in "batch" systems, aiming to obtain higher final yield of astaxanthin. The work was divided into two phases. First, to the control (containing only H. pluvialis) it was applied two combinations of stress factors to induce cellular synthesis of astaxanthin by the microalgae, to define the method of stress to be applied in the next phase. The cells of H. pluvialis were submitted to stress by high light intensity (seven times the original) or the same light stress combined with CO2 supplementation (4%) in the aeration provided in the exponential growth phase. We evaluated, among other factors of cellular response, the final concentration of astaxanthin in the biomass after 10 days of stress. The pigment concentrations were 4.15 and 17.66 mg g-1, respectively, for cultures submitted to light stress and to those who received the same light stress add with the increase of C / N ratio in the culture medium. So, for the next experimental phase, the combination... (Complete abstract click electronic access below) / Doutor

Les biofilms ont longtemps été décrits comme des organisations évolutives de microorganismes, attachés à une surface et englués dans une matrice contenant, entre autre, des polysaccharides. En partant de ce constat BioFilm Control a souhaité cribler des microorganismes pour la production d’exopolysaccharides, en utilisant le BioFilm Ring Test® (BRT). Le principe repose sur la coincubation de microorganismes avec des particules magnétiques en microplaque. Les particules sont plus ou moins attirées par un aimant en fonction du stade d’organisation du biofilm. En se formant, il piège, dans sa matrice visqueuse, les particules qui perdent leur mobilité. Celle-ci est révélée par une aimantation qui provoque l’apparition d’un spot (pas de biofilm) ou non (biofilm). Une analyse d’images quantifie ce processus et permet de le standardiser. La démarche a consisté dans un premier temps à vérifier le comportement de microorganismes modèles producteurs de polysaccharides (bactéries et microalgues) avec le BRT. L’étude a été étendue au criblage d’une banque de lactobacilles. Les résultats inattendus ont orienté l’étude vers l’analyse du rôle exact des polysaccharides et plus généralement de l’implication des macromolécules dans la structuration du biofilm. Pour cela, la dégradation séquentielle de chaque famille macromoléculaire a été réalisée via des enzymes dépolymérisantes sur les biofilms de Leuconostoc mesenteroides et Bacillus sp. Au regard des résultats obtenus, l’utilisation du BRT a été étendue à la caractérisation qualitative et quantitative d’enzymes de dégradation de polysaccharides. / Biofilms were described for a long time as evolutionary structures elaborated by microorganisms, fixed on a surface and maintained in a polysaccharidic matrix. From this assessment, BioFilm Control chose to screen microorganisms for their capacity to produce exopolysaccharides (EPS), using theBioFilm Ring Test® (BRT). The principle is the co-incubation of magnetic particles with microbial culture on microplates. The mobility of particles depends on the stage of biofilm formation. During this formation, particles are trapped in the matrix and loose their mobility. Revelation is induced by magnet which causes a spot in the absence of biofilm. The pictures analysis quantifies this phenomenon and standardizes different results. This approach was realised, at first step, by the test of EPS-producing bacteria or microalgae with the BRT. The study was extended to the screening of a lactobacilli collection. Unexpected results guided the research toward the understanding of the role of macromolecules in biofilm structuring. To study their implication, sequential enzymatic degradation has been achieved for each macromolecular family of Leuconostoc mesenteroïdes and Bacillus sp. biofilms. Using the results, BRT was then appreciated as a suitable method to detect and quantify polysaccharide degrading enzymes.

The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain high amounts of free fatty acids, which are unsuitable for base-catalyzed transesterification. Herein an approach is described that efficiently accomplishes the simultaneous esterification and transesterification of both free fatty acids and triglycerides found in low-cost oils. The approach utilizes an acid catalyst and longer-chain alcohols to improve biodiesel yields from oils high in free fatty acids. Microalgae are a promising biodiesel feedstock, due to its high lipid productivity and its ability to be cultivated using resources, land and water, unsuitable for agriculture. As part of this work, reaction conditions were optimized for the direct (or in situ) transesterification of algal biomass to biodiesel. This approach accomplishes the simultaneous extraction and conversion of the total lipids from microalgae and results in increased yields compared to extraction followed by conversion. The use of this process to effectively produce biodiesel from wet algal biomass is also discussed. Wax esters are a class of oleochemicals that can be used for a wide range of applications in diverse industries. The chemical composition of native wax esters from the bacterium Marinobacter aquaeolei was determined. It was found that including small alcohols in the growth medium resulted in the in vivo formation of esters similar to biodiesel. All of the proteins involved in the wax ester biosynthetic pathway are not known. The cloning, purification, and characterization of a putative fatty aldehyde reductase from M. aquaeolei, believed to be involved in the production of wax esters, is reported. Finally, the expression of a ws/dgat (wax ester synthase) gene from M. aquaeolei in the cyanobacterium Synechocystis sp. PCC 6803 is discussed as an approach to producing biodiesel in vivo from sunlight and CO2.

Though usually a nuisance in swimming pools and ponds, algae has the potential to be a valuable commodity for use as food and fuel. But before algae butter and biofuel become commonplace, issues with harvesting and storing this new crop need to be overcome. Though there has been ample research into how to grow and use algae, scientists have spent little time figuring out what to do after you pull it out of the water and before you eat it (or turn it into biodiesel). Algae, like all food products, starts to spoil as soon as it is harvested. This study looked at three methods of preserving algae, freezing, drying, and pasteurization. Freezing is a good method for preserving fats and proteins, but it is expensive to freeze tons of algae. Freezing and thawing destroyed the algal cells, producing a soupy mixture that may cause complications for processing into foods or fuels. Drying was able to preserve the fats in algae, but only if it was dried just the right amount, about as dry as cheese or ham. Pasteurization was able to prevent the oils in the algae from going rancid by inactivating a protein in the algae that causes the oil to spoil rapidly. Overall, this research is an initial step in finding a process to produce a shelf stable algal commodity, opening the door to new and valuable products for human use.

Phototropic microalgae have a large potential for producing valuable substances for the feed, food, cosmetics, pigment, bioremediation, and pharmacy industries as well as for biotechnological processes. Today it is estimated that the microalgal aquaculture worldwide production is 5000 tons of dry matter per year (not taking into account processed products) making it an approximately $1.25 billion U.S. per year industry. In this work, several spectroscopic techniques were utilized for the investigation of microalgae cells. Specifically, photondensity wave spectroscopy was applied as a technique for the on-line observation of the culture. For effective evaluation of the photosynthetic growth processes, fast and non-invasive sensor systems that analyze the relevant biological and technical process parameters are preferred. Traditionally, the biomass in a photobioreactor is quantified with the help of turbidimetry measurements, which require extensive calibration. Another problem frequently encountered when using spectral analysis for investigating solutions is that samples of interest are often undiluted and highly scattering and do not adhere to Beer-Lambert's law. Due to the fluorescence properties of chlorophyll, fluorescence spectroscopy techniques including fluorescence lifetime imaging and single photon counting could be applied to provide images of the cells as well as determine the effects of excitation intensity on the fluorescence lifetime, which is an indicator of the condition of the cell. A photon density wave is a sinusoidally intensity-modulated optical wave stemming from a point-source of light, which propagates through diffuse medium and exhibits amplitude and phase variations. Light propagation though strongly scattering media can be described by the P1 approximation to the Boltzmann transport equation. Photon density wave spectroscopy enables the ability to differentiate between scattered and absorbed light, which is desired so that an independent determination of the reduced scattering and absorption coefficients can be made. The absorption coefficient is related to the pigment content in the cells, and the reduced scattering coefficient can be used to characterize physical and morphological properties of the medium and was here applied for the determination of the average cell size. / Phototropische Mikroalgen besitzen ein großes Potential für die Herstellung von wertvollen Substanzen sowohl für die Futtermittel-, Lebensmittel-, kosmetische und pharmazeutische Industrie, als auch für die Farbstoffsynthese. Heutzutage werden schätzungsweise 5000 Tonnen Mikroalgen Trockensubstanz pro Jahr mit einem Jahresumsatz von 1,25 Mrd. US-Dollar produziert. In dieser Arbeit wurden diverse spektroskopische Untersuchungsmethoden für die Betrachtung der Zellen verwendet. Die Photonendichtewellenspektroskopie (PDW) fand dabei insbesondere bei der on-line Beobachtung der Zellen Anwendung. Voraussetzungen für die effektive Beobachtung von photosynthetischen Wachstumsprozessen sind schnell und nicht-invasiv arbeitende Sensoren. Normalerweise wird die dabei zu untersuchende Biomasse in einem Photobioreaktor mittels Trübungsmessungen quantifiziert. Dies setzt jedoch eine sehr aufwendige Kalibration voraus. Da diese Proben zusätzlich meist in unverdünnter Form vorliegen, streuen sie stark und folgen daher nicht dem Lambert-Beer'schen Gesetz. Aufgrund der Fluoreszenzeigenschaften des Chlorophylls können fluoreszenzspektroskopische Methoden wie fluorescence lifetime imaging microscopy (FLIM) und time correlated single photon counting (TCSPC) angwendet werden. Diese Methoden werden dabei für die visuelle Darstellung der Zellen und für die Messung der Fluoreszenzlebenszeit genutzt. Eine Photondichtewelle ist eine sinusförmig intensitätsmodulierte optische Welle, die sich, ausgehend von einer punktförmigen Lichtquelle, durch das untersuchte Medium ausbreitet, wobei sich Phase und Amplitude der Welle verändern. Die Ausbreitung der Welle wird von der P1-Näherung der Boltzmann-Transport-Gleichung beschrieben. Mittels PDW kann zwischen streuenden und absorbierenden Eigenschaften von trüben Probe unterschieden werden. Dies erlaubt die Absolutbestimmung des Absorptions- und reduzierten Streukoeffizienten, die für die Qualifizierung der Probe, insbesondere der Teilchengrößenbestimmung, herangezogen werden.

The goal of this study was to characterize the nutritional contribution of microalgae to white-legged shrimp and optimize fish meal (FM) and fish oil (FO) inclusion levels in their diets in the presence of microalgae. Phytoplankton composition was first determined in a typical Peruvian intensive commercial shrimp farm and in a semi-closed greenhouse-covered reservoir. A predominance of 76.3% cyanobacteria was observed for most of 9 months in all shrimp ponds. However, with the fertilization program in a reservoir tank, 60.7% diatoms and 22.8% cyanobacteria predominated. Thus, with the imposed fertilization regimen, the microalgae composition was manipulated to be different than that in commercial shrimp ponds. The microalgae composition was then evaluated along with different dietary levels of FM and squid meal (SM) in a feeding trial to evaluate the potential of phytoplankton to reduce FM and SM levels in shrimp feeds. Six diets were formulated to contain either 5, 10 or 20% SM combined with either 6.5 or 12% FM. Dietary effects on growth and survival were compared in a "clear-water system" (CWS) and a "green-water system" (GWS). Results suggest that 6.5% FM and 5% SM can be used as a cost-effective combination in feeds for shrimp. The effects of different dietary levels of FO and soybean lecithin (LT) on shrimp growth in CWS and GWS were evaluated in another feeding trial to determine if dietary phospholipids and phytoplankton increase the availability of essential fatty acids (EFAs) to shrimp. Six diets were formulated to contain 1, 2 or 3% FO combined with either 1 or 4% LT. Shrimp fed diets containing 1% LT and 1% FO in both systems had significantly lower weight gain and higher feed conversion ratio. Cephalothorax lipids and phospholipids were higher in shrimp fed diets containing 4% LT. Inclusion of 4% LT increased the availability of EFAs, and could contribute to reduce the FO in shrimp diets. The contribution of phytoplankton to shrimp weight gain, varied from 38.8 to 60.6%. This study demonstrated that cost-effective diets could be formulated with reduced inclusion levels of FM and FO considering the contribution of microalgae to the nutrition of shrimp.

As part of a research project aiming to develop and evaluate a hydroponic system for wastewater treatment in Sweden, extended nutrient removal by microalgae was tested. The hydroponic/microalgal wastewater treatment system was built in a greenhouse in order to improve growth conditions for plants and algae. Studies on the treatment step with microalgae showed that phosphorus removal could be successfully accomplished owing to the cmbined effect of phosphorus assimilation and biologically mediated chemical precipitation of calcium phosphates. This precipitation was mainly induced by the increased pH in the algal cultures, and the pH increase was in turn a result of the inorganic carbon assimilation by the algae. The results showed that the algal growth was mainly light limited which resulted in higher algal biomass density and also lowe residual nutrients in the water at longer hydraulic retention times (HRT). In contrast the phosphorus removal rate was load limited, i.e. shorter HRT gave higher removal rates. This load dependency was due to the chemical precipitation, whereas the phosphorus assimilation was dependent on algal growth. Furthermore, results from an intensive study during summer showed that culture depths of 17 cm gave higher removal efficiencies (78% - 92%) than cultures of 33 cm (66% - 88%). On the other hand, the removal rate per area was higher in the deeper cultures, which implies that these may be preferred if area is of concern. Nitrogen removal was achieved mainly by the assimilation of nitrate to algal biomass, and removal efficiencies of around 40% (nitrate) could be reached for most parts of the year although the nitrogen removal performance was quite uneven. Up to 60% - 80% could however be reached during summer in the shallow cultures. A net removal in total nitrogen of up to 40% was observed in the shallow cultures during summer, which was most probably a consequence of grazing zooplankton and subsequent urea excretion and ammonia volatilisation as a reslt of the high pH values. Over the year, there were large fluctuations in algal growth and removal efficiency as a result of the seasonal variations in light and tempeature. During winter, phosphorus removal efficiencies lower than 25% were observed in the shallow tanks and lower than 10% in the deep tanks. Additional illumination during winter improved the phosphorus removal in the shallow cultures but did not have a significant efect on the deep cultures. Such additional illumination increases the total energy demand of the system, and hence alternative methods for phosphorus removal during winter would probably be more economical unless the algal biomass roduced had great commercial value. / QC 20100907

Immobilization of photosynthetic cultures has been used to generate biofuels and high value compounds through direct conversion of CO2 and water using sunlight. Compared with suspended cultures, immobilized bacteria can achieve much higher densities resulting in greater areal productivity. Limitations exist however, on the density that can be reached without compromising access to light and other nutrients. In this thesis an optofluidic approach to overcoming the challenge of light delivery to high density cultures of cyanobacteria is described and proof of concept experiments presented. This approach uses optical waveguides to deliver light to cells through bacterial interaction with the evanescent field and is tailored to meet each cell's need for light and nutrients. Experiments presented here demonstrate biofilm proliferation in the presence of evanescent fields. Illumination of surfaces by surface plasmon enhanced evanescent fields is also shown to be an effective and potentially useful technique to grow biofilms within optofluidic architectures.

Benthic microalgal community composition on an ephemerally submerged sandflat at East Beach, Galveston Island, Texas was studied to determine the spatial and temporal variability of total biomass and community composition and its responses to experimental manipulations of two environmental factors (salinity and nutrients). Four field studies were conducted between August 2004 and February 2005. The community consisted of two major algal groups, diatoms, and cyanobacteria with two less abundant groups, green algae, and phototrophic bacteria. Spatial variability showed that patch sizes of 12 - 25 m were detected over larger scales with smaller scale (cm) patches of approximately 28 - 201 cm^-2 contained within the larger patches. The second study examined the spatio-temporal variability of BMA over a 21-month period in a 1,000 m^2 area. Sampling location and date explained a significant amount of the variability in the abundances of algal groups, which were positively correlated with the water content of the sediments and negatively correlated with temperature (sediment and water). All of the algal groups showed a seasonal pattern with higher abundances measured in the winter months and lower abundances found during the summer. BMA biomass (100 mg Chl a m^-2 or greater) maxima occurred at temperatures less than 22 degrees C and sediment water content greater than 15% (g water g sediment^-1). BMA response to different salinities and nutrient (N+P) amended sediments was assessed in four bioassays conducted over a 6-month period (Aug. 2004, Oct. 2004, Dec. 2004, and Feb. 2005). In the salinity study, the treatments that were either 100% or partially diluted with deionized water had the lowest BMA biomass over all. Chlorophyll a and fucoxanthin were significantly affected by salinity with higher abundances found in salinities that averaged 15 with a preference for salinities greater than 22. Chlorophyll b was affected by salinity with higher abundances measured in the treatments with lowest salinity (DL and DI); and was affected by the time of year. This would suggest that this algal group prefers an environment with salinity less than 2 but can easily adapt to environments with higher salinities. BMA abundances were not significantly affected by the nutrient amended sediment, but were significantly affected by stations with higher water content, and during the cooler months (Dec. 2004 and Feb. 2005).