Growth media recycling during algae cultivation is necessary to increase the efficiency and reduce the cost of biofuel production from algae feedstocks. Without recycling media, the cost of algae based biofuel production would be prohibitively high and large scale algae based biofuel production would not be economically viable. The ratio of media recycled to media wasted assumed for algae farms is generally calculated to maintain salt concentrations below growth inhibitory levels, ignoring the influence of secondary metabolites which might decrease productivity. Secondary metabolites, which include allelopathic or auto-inhibitory biological contaminants, might lead to the accumulation of growth-inhibiting compounds in recycled media used in algae production. Chlorella sorokiniana (strain DOE1412) was a leading algae biofuel feedstock candidate and has not previously been evaluated for inhibitor production. To test the effects of water recycling on the growth of DOE1412, media was recycled through multiple rounds of algae cultivation. DOE1412 was grown in modified BG11 culture media until reaching the end of linear growth phase, at which point the biomass was removed, nutrients replenished to their initial concentrations, and the recycled culture media used for a subsequent round of growth. The culture media was recycled through five rounds of growth with cultures grown on recycled media compared to controls grown on freshly prepared growth media. Biomass density was monitored via optical density and the specific and productivity growth rates were used to quantify the extent of inhibition. Exploratory work was performed with the goal of identifying potential inhibitory substances produced by DOE1412 during cultivation. Samples of recycled media were analyzed for polyunsaturated fatty acids which have been demonstrated to be inhibitory. The carbohydrates content of used media was analyzed to assess the amount of organic materials shed by DOE1412 into recycled media during growth. The log phase growth rate (day-1) of DOE1412 was inhibited by 3±2%, 8±1%, 10±2%, and 18.6±0.9% when grown in media recycled 1-4 times, respectively, with a 99% level of confidence that inhibition was observed in each round of regrowth. The productivity growth rate (OD750/day) of DOE1412 was not inhibited in media recycled 1-3 times. The productivity growth rate of DOE1412 was inhibited by 13±3% when grown in media recycled 4 times with a 99% level of confidence that inhibition was observed. Zinc was found to accumulate in the recycled media to potentially toxic levels (>0.09 mg/L), therefore it is uncertain if the observed inhibition was due to an accumulation of inhibitory secondary metabolites or the accumulation of zinc. Two inhibitory polyunsaturated acids, linoleic and linolenic acid, were identified in media recycled 4 times. The carbohydrate content of recycled media fluctuated between 8-10% of total fixed carbon in media recycled 1-3 times and increased to 18% in media recycled 4 times. However, changes observed in media recycled 4 times may have been due to improper storage of used media.

No description available.

No description available.

Synchronous cultures of a high temperature strain of Chlorella pyrenoidosa have been used to determine the activity of the TMP kinase during cellular development. It was observed that the apparent enzyme activity was closely correlated with the rate of DNA biosynthesis. During the period of nuclear division, when DNA synthesis is at its maximum apparent TMP kinase activity is highest. The maximum in enzyme activity, however, slightly precedes the peak in DNA synthesis. These results support the hypothesis that TMP kinase activity is a factor controlling the rate of DNA biosynthesis. It appeared that the dramatic shifts in the level of TMP kinase activity were a result of variations in the rate of enzyme synthesis rather than control of enzyme activity by small molecule inhibitors or activators. / Ph. D.

The factors which regulate enzyme activity in the growing cell have been the subject of active research during the past decade and are generally considered under the heading of "metabolic control". At least three control parameters must be considered as regulating the active level of a given enzyme: control of enzyme synthesis at the gene level, control of enzyme activity by small molecule metabolites once the enzyme has been synthesized, and control of enzyme activity imposed by its structural orientation within the cell and/or its organelles (which would affect permeability of substrates, cofactors, etc.). It can be hypothesized that periodism in the intracellular level of a given metabolite must be accompanied by periodism in the active level of one or more of the enzymes responsible for the synthesis or breakdown of this metabolite. Synchronized cultures of microorganisms afford a unique tool for studying periodic changes in the intracellular levels of metabolites during cell growth. The studies presented in this thesis were designed to elucidate the relationships between the activity of Aspartate Transcarbamylase and factors which affect and are affected by the activity of this enzyme in synchronized cultures of Chlorella pyrenoidosa. Aspartate Transcarbamylase, the first enzyme in pyrimidine biosynthesis, has been reported to be controlled by a phenomenon known as " product-inhibition". This enzyme was located in the soluble supernatant of a 100,000 x g preparation of sonicated C. pyrenoidosa ceils. The pH optimum (9.2), temperature optimum (approx. 37°), and stability characteristics of this enzyme from this organism are reported. Aspartate Transcarbamylase when measured during two consecutive synchronous growth cycles in C. pyrenoidosa under continuous illumination was found to increase at alternating exponential rates in each growth cycle. When the increase in Aspartate Transcarbamylase was expressed in its logarithmic form, a plot with linear segments was obtained, each segment having a different slope. The rate of increase of Aspartate Transcarbamylase activity during early daughter cell development changes (increases) at the 5th h. The 5th through 9th h of cell growth, where Aspartate Transcarbamylase activity is increasing at an accelerated rate, corresponds to the premitotic and nuclear division stages in the cell. Correlated with this period of increased Aspartate Transcarbamylase activity (5th through 9th h) there is an increase in the rate of accumulation of RNA and DNA concurrent with increase in the rate of accumulation of intermediates in the acid-soluble, Norit-A adsorbable-P pool (which contains the nucleotide-P fraction). At. approximately the 9th h of cell growth there is a decrease in the rate of increase of Aspartate Transcarbamylase activity. Evidence is presented indicating that the depression of activity during this period is at least partly due to the presence of Norit-A adsorbable compound(s) present in the cell (pyrimidine nucleotides, the principal inhibitors of this enzyme, would be Norit-A adsorbable). At the 8th h of cell growth ( 1h prior to the period of depressed Aspartate Transcarbamylase activity) the acid-soluble, Norit-A adsorbable-P pool reaches a maximum value (as % of total cellular-P). These data together with inhibition studies with a variety of nucleoside mono-, di-, and tri-phosphates suggest that pyrimidine nucleotides may be factors regulating Aspartate Transcarbamylase activity during cellular development. / Ph. D.

The ammonium inducible nicotinamide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana has been purified 260-fold to homogeneity. Depending on the technique used, the native enzyme appeared to have a molecular mass of 290,000 to 400,000 daltons and to be composed of subunits with an identical molecular weight of 58,000. Differences in the molecular weight of the native enzyme, as determined by sedimentation equilibrium, Sephadex G-200 gel filtration and gradient polyacrylamide gel electrophoresis, indicate that the native enzyme may be elliptical in shape. The amino acid composition of the enzyme is high in glycine, glutamate, and asparate. Moreover, the arginine to lysine ratio is similar to those measured in other glutamate dehydrogenases. The Nterminal amino acid is unavailable to dansylation. All six cysteines in the enzyme are in the free sulfhydryl form. The enzyme is very specific for the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate and has less than 0.5 percent of maximal activity, using the oxidized and reduced forms of nicotinamide adine dinucleotide. With low concentrations of the substrates, no cooperativity was seen; however severe substrate inhibition was observed with a-ketoglutarate. Antiserum produced to the subunits of the enzyme yielded a single precipitin band against purified enzyme in Ouchterlony double diffusion analysis. "Rocket" immunoelectrophoresis has been used to quantify the amount of antigen present in samples of the purified enzyme. / Master of Science

M.S.

M.S.

As microalgas têm sido objeto de diversos estudos visando-se à produção de biocombustíveis ou outros produtos de interesse dentro do conceito de biorrefinaria. Na produção de biocombustíveis, as microalgas têm sido utilizadas por apresentarem elevada produtividade em lipídeos e carboidratos. Para viabilização de seu uso como matéria prima, é fundamental o desenvolvimento de processos com elevada produção de biomassa. Os fatores que influenciam no crescimento e composição da biomassa microalgal incluem a seleção do biorreator, a concentração e natureza da fonte de nitrogênio e o tipo de cultivo com relação às fontes de carbono e energia (autotrófico, heterotrófico ou mixotrófico). Neste trabalho, a microalga Chlorella minutissima 26a foi cultivada em fotobiorreatores do tipo tanque de bolhas em modo batelada ou airlift de tubos concêntricos em modo contínuo. Em cultivo autotrófico, estes fotobiorreatores foram empregados para avaliação da influência da concentração de nitrato de sódio no meio de cultivo no crescimento microalgal, na composição química da biomassa e na produtividade em lipídeos, carboidratos, proteínas e biomassa seca. Foi analisado também o uso de lixiviado proveniente de aterro sanitário na composição do meio de cultivo em fotobiorreator airlift em processo mixotrófico contínuo, promovendo concomitante biorremediação do lixiviado empregado. Os resultados demonstraram que a concentração de nitrato no meio de cultivo influenciou o crescimento e a composição química da biomassa e, consequentemente, os valores de produtividade obtidos. Nos ensaios usando fotobiorreator de tanque de bolhas em batelada, a variação da concentração de nitrato de sódio no meio de 37,5 mg L-1 para 150 mg L-1 promoveu aumento da produtividade em lipídeos e carboidratos, com valores máximos de 105,2 ± 1,7 mg L-1 d-1 e 36,7 ± 0,6 mg L-1 d-1, respectivamente, obtidos com a maior concentração de nitrato avaliada. No cultivo usando o fotobiorreator airlift em processo contínuo, o aumento da concentração de nitrato de sódio de 75 mg L-1 para 225 mg L-1 resultou nos maiores valores de produtividade média no estado estacionário para biomassa, lipídeos, proteínas e carboidratos, os quais foram, respectivamente, de 188,6 ± 11.2 mg L-1 d-1, 92,8 ± 5,5 mg L-1 d-1, 37,7 ± 2,2 mg L-1 d-1 e 29,1 ± 1,7 mg L-1 d-1. Neste caso, quando a concentração de nitrato de sódio aumentou de 75 mg L-1 para 150 mg L-1, observou-se aumento no teor de lipídeos e redução no teor de carboidratos da biomassa, sem modificação composicional apreciável quando a concentração de nitrato de sódio foi elevada para 225 mg L-1. O emprego do lixiviado diluído como meio de cultivo da Chlorella minutissima 26a resultou em elevados valores de produtividade em lipídeos, carboidratos, proteínas e biomassa, correspondendo a valores máximos de 232,0 ± 7,6, 95,3 ± 5,2, 33,4 ± 2,1 e 69,2 ± 3,0 mg L-1 d-1, respectivamente, dependendo da concentração de lixiviado no meio. O cultivo microalgal em lixiviado diluído resultou ainda em remoção de até 92,8% da demanda química de oxigênio, 90,5% do carbono orgânico total e 100% do nitrato presente no meio. Além disso, metais presentes foram removidos do meio durante o cultivo, resultando na remoção de até 63%, 72%, 100% e 67% para Cr, Fe, Al e Ba, respectivamente. O fotobiorreator airlift de tubos concêntricos demonstrou grande potencial para o cultivo da microalga C. minutissima em processo contínuo, incluindo a possibilidade de uso de lixiviado diluído na composição do meio para cultivo mixotrófico com sua concomitante biorremediação. / Microalgae have been the subject of different studies aimed at the production of biofuels or other products of interest within the concept of biorefinery. In the production of biofuels, microalgae have been used because they present high productivity in lipids and carbohydrates. In order to enable its use as a raw material, the development of processes with high biomass production is fundamental. Variables influencing the growth and composition of microalgal biomass include the selection of the bioreactor, the concentration and kind of the nitrogen source and the type of cultivation with respect to carbon and energy sources (autotrophic, heterotrophic or mixotrophic). In this work, the microalgae Chlorella minutissima 26a was cultivated in photobioreactors of two different kinds: bubble tank in batch mode or airlift of concentric tubes in continuous mode. In autotrophic cultivation, these photobioreactors were used to evaluate the influence of sodium nitrate concentration in the culture medium on microalgal growth, chemical composition of biomass and productivity in lipids, carbohydrates, proteins and dry biomass. The use of landfill leachate was also analyzed as a component of the culture medium in continuous airlift photobioreactor in a mixotrophic process, promoting concomitant bioremediation of the used leachate. The results showed that the concentration of nitrate in the culture medium influenced the growth and chemical composition of the biomass and, consequently, the productivity values obtained. In the assays using batch bubble tank photobioreactor, the variation in the sodium nitrate concentration in the medium from 37.5 mg L-1 to 150 mg L-1 promoted an increase of the productivity in lipids and in carbohydrates, with maximum values of 105.2 ± 1.7 mg L-1 d-1 and 36.7 ± 0.6 mg L-1 d-1, respectively, obtained with the highest evaluated nitrate concentration. In the cultivation using continuous airlift photobioreactor, the increase in sodium nitrate concentration from 75 mg L-1 to 225 mg L-1 resulted in higher values of average productivity in steady-state for biomass, lipids, proteins and carbohydrates, which were, respectively, of 188.6 ± 11.2 mg L-1 d-1, 92.8 ± 5.5 mg L-1 d-1, 37.7 ± 2.2 mg L-1 d-1 and 29.1 ± 1.7 mg L-1 d-1. In this case, when the sodium nitrate concentration increased from 75 mg L-1 to 150 mg L-1, there was an increase in lipid and a reduction in the carbohydrate content of the biomass, with no appreciable compositional modification when the sodium nitrate concentration was increased to 225 mg L-1. The use of diluted leachate as a culture medium of Chlorella minutissima 26a resulted in high productivity values in lipids, carbohydrates, proteins and biomass, corresponding to maximum values of 232.0 ± 7.6, 95.3 ± 5.2, 33. 4 ± 2.1 and 69.2 ± 3.0 mg L-1 d-1, respectively, depending on the landfill leachate concentration in the medium. The microalgal cultivation in diluted leachate also resulted in the removal of up to 92.8% of the chemical oxygen demand, 90.5% of the total organic carbono and 100% of the nitrate present in the medium. In addition, present metals were uptake from the medium during cultivation, resulting in a removal of up to 63%, 72%, 100% and 67% for Cr, Fe, Al and Ba, respectively. The airlift photobioreactor of concentric tubes demonstrated great potential for the cultivation of C. minutissima microalgae in a continuous process, including the possibility of using landfill leachate diluted in the medium composition for mixotrophic cultivation with its concomitant bioremediation.

Des cultures de Chlorella vulgaris ont été réalisées en triplicata sur de longues périodes (15-30 jours) en autotrophie et en mixotrophie afin d’étudier l’effet de Mg2+ sur les microalgues et déterminer sa concentration dans la biomasse au cours de la croissance. Différentes concentrations ont été testées (de 8.9 à 465 mg L-1). Ceci a montré que le Mg2+ n’est pas toxique pour les algues à des concentrations élevées provoquant habituellement l’inhibition de la croissance dans le cas d’autres ions divalents (métaux lourds). Des bilans matière ont été établis et vérifiés avec une précision satisfaisante (écart moyen de 90%). La quantité de Mg2+ bioaccumulée augmente avec la concentration initiale de Mg2+ du milieu de culture. Une saturation en Mg2+ intracellulaire a été observée à partir d’environ 45 mg de Mg2+ L-1. Typiquement, la répartition entre le Mg2+ extracellulaire et intracellulaire est de 18% contre 51% dans le bioréacteur (5L) pour une concentration initiale de 16 mg L-1. En mixotrophie (addition de 10 g L-1 de glucose), une production de biomasse significativement plus importante et plus rapide que celle en autotrophie a été observée. En revanche, la quantité de Mg2+ accumulée dans les cellules est plus importante en autotrophie qu’en mixotrophie. Des mesures de chlorophylle a, de nitrate et de glucose ont été également réalisées. Afin de comprendre la cinétique relativement complexe de bioaccumulation du Mg2+, un modèle cinétique original a été élaboré couplant transfert (externe, membrane, interne) et réactions (ad/absorption) en milieu hétérogène. Le modèle a été validé expérimentalement. Il permet de rendre compte au mieux de l’ensemble des résultats obtenus. / Chlorella vulgaris cultures were grown in triplicate over 15-30 days under autotrophic or mixotrophic conditions in order to study Mg2+ uptake and accumulation into the biomass. The concentrations of Mg2+ tested were 8.9 to 465 mg L-1. The results showed that Mg2+ is not toxic to C. vulgaris even at 465 mg L-1 which is not the case for other divalent ions (especially heavy metals). The mass balances for Mg2+ accumulation were determined for the cultures and were confirmed to an average of 90%. Beyond 46.1 mg of Mg2+ L-1 in the culture medium, the cells became saturated at 4 mg of Mg2+ absorbed per g of dry biomass. In the bioreactor (5L), at an initial Mg2+ concentration of 16 mg L-1, the cells accumulated 69% of the initial concentration, in which 18% was adsorbed and 51% absorbed. The chlorophyll a, nitrate and glucose concentrations were measured during the experiments. Under mixotrophic conditions (glucose 10 g L-1), a greater and faster biomass production was obtained than under autotrophic conditions. The Mg2+ bioaccumulation was higher in the autotrophic rather than the mixotrophic phase. In order to understand the kinetics of Mg2+ uptake, an original kinetic model was developed coupling the transfer and reaction phenomena in heterogeneous media. This model was confirmed experimentally.