Optimising illumination is essential for optimising the growth of phototrophic cells and their production of desired metabolites and/or biomass. This requires appropriate modulation of light and other key inputs and continuous online monitoring of their metabolic activities. Powerful non-invasive systems for cultivating heterotrophic organisms include shake flasks in online monitoring units, but they are rarely used for phototrophs because they lack the appropriate illumination design and necessary illuminatory power. This study presents the design and characterisation of a photosynthetic shake flask unit, illuminated from below by warm white light-emitting diodes with variable light intensities up to 2300 μmol m-2 s-1. The photosynthetic unit was successfully used, in combination with online monitoring of oxygen production, to cultivate Arthrospira platensis. In phototrophic growth under continuous light and a 16 h light/8 h dark cycle (light intensity: 180 μmol m-2 s-1), the oxygen transfer rate and biomass-related oxygen production were - 1.5 mmol L-1 h-1 and 0.18 mmol O2 gx-1 h-1, respectively. The maximum specific growth rate was 0.058 h-1, during the exponential growth phase, after which the biomass concentration reached 0.75 g L-1.

Des travaux préliminaires réalisés en fiole d'Erlenmeyer ont montré que l'environnement hydrodynamique, caractérisé par la puissance dissipée volumique (P/V) et le coefficient de transfert en oxygène (kLa), jouait un rôle important lors du procédé de production de pristinamycines par Streptomyces pristinaespiralis (Mehmood, 2011). L'objectif de ce travail est donc d'étudier l'influence de ces deux phénomènes dans des bioréacteurs mécaniquement agités (STR), largement utilisés à l'échelle industrielle, et de type gazosiphon. Dans un premier temps, une description de l'environnement hydrodynamique global a été réalisée en STR. En ce qui concerne le bioréacteur gazosiphon, celui-ci a été conçu et dimensionné spécifiquement pour ce travail. Une caractérisation des écoulements dans ce bioréacteur a ensuite été réalisée par simulation numérique des écoulements. En appliquant les mêmes conditions hydrodynamiques que celles étudiées lors de culture en fioles d'Erlenmeyer, les performances en terme de croissance et de production de pristinamycines ont toujours été moindres en STR et en gazosiphon qu'en fiole. Ceci démontre que P / V, kLa mais également la dissipation maximum, ne constituent pas les bons paramètres d'extrapolation. Par contre, les performances mesurées semblent être reliée aux variations de formes morphologiques observées (présence et taille des pelotes) et à la physiologie des cellules au sein de ces structures. De façon surprenante, au cours de ce travail, le déclenchement de la production de pristinamycines a quasiment toujours été obtenu lors de la phase de croissance de S. pristinaespiralis. Nos travaux n'ont pas permis de mettre en évidence la raison de ce phénomène. Celui-ci est certainement la conséquence de plusieurs paramètres qui restent encore à préciser / Previous results performed in Erlenmeyer flasks have shown that the hydrodynamics, characterized by power dissipation per unit of volume (P/V) and volumetric oxygen mass transfer coefficient (kLa), impacted the production of pristinamycins by Streptomyces pristinaespiralis (Mehmood, 2011). The aim of this work is then to study the influence of these two parameters in a stirred tank bioreactor (STR), widely used in industry, and in an airlift bioreactor. This last bioreactor has been designed specifically for this work. In a first part, the hydrodynamic environment was described in STR and the fluid flows were simulated by computational fluid dynamics (CFD). Using the same hydrodynamic conditions in STR and in airlift bioreactor than in flasks, the process performance (bacterial growth and pristinamycin production) were always lower in STR and airlift bioreactor. This demonstrates that P / V, kLa and also maximum dissipation were not pertinent scale-up criteria for the pristinamycin production from flask to STR or airlift bioreactor. On the contrary, the determined performances seemed to be related to the changes in bacterial morphology (presence and size of pellets) and to the physiology of the cells inside these structures. Surprisingly, during this work, the initiation of the pristinamycin production occurred almost always during the growth phase of S. pristinaespiralis. This phenomenon was probably due to the conjunction of several parameters which remain to identify

Le but principal de cette étude était d'évaluer et de mieux comprendre l'impact de la vitesse superficielle de gaz et des propriétés de boues activées (BA), sur leur comportement rhéologique et le transfert de l'oxygène dans des bioréacteurs.Tout d'abord, la rhéologie des BA a été évaluée à l'aide d'un rhéomètre tubulaire, conçu et construit dans ce travail. Des mesures rhéologiques ont été effectuées avec des BA provenant de cinq stations d'épuration (STEP) et avec des concentrations en MES comprises entre 2.3 et 10.2 g L-1. Selon ces résultats, la rhéologie des BA est significativement déterminée par la concentration en matière en suspension (MES) mais d'autres caractéristiques liées à leur origine, tel que la taille, la cohésion et la densité du floc, peuvent aussi influencer la viscosité apparente des boues. Basé sur les rheogrames expérimentaux, le modèle rhéologique issu de cette étude est comparé à des modèles rhéologiques existants.Deuxièmement, le transfert d'oxygène a été évalué dans une colonne à bulles (Hc=4.4 m, Dc=0.29m) installée dans deux STEP: une installation classique et un bioréacteur à membrane. La colonne, alternativement équipée d'un diffuseur fines ou grosses bulles (FB, GB), a été alimentée en continu avec des BA extraites du réacteur d'aération, ou de la boucle de recirculation ou du réacteur membranaire. Pour des MES comprises entre 3.0 et 10.4 g L-1, le coefficient kLa a étéplus faible dans les BA que dans l'eau propre et encore réduit avec une augmentation des MES. Cette diminution est en partie attribuable à la réduction observée de la rétention de gaz (εG), associée à une augmentation de la viscosité apparente des boues, celle-ci entrainant une réduction de l'aire interfaciale spécifique (a) due à la coalescence de bulles et à la formation de bulles plus grosses. Aussi, la concentration des tensioactifs non ioniques, a montré un effetnégatif sur le coefficient kLa lors des tests d'oxygénation effectués en aération FB et faibles concentrations en MES. Cet impact n'a pas été observé dans des conditions d'aération GB, ce qui a été expliqué par le taux de renouvellement d’interface plus élevé généré par ces dernières. Enfin, le taux de cisaillement moyen exercé par l'essaim de bulles dans la colonne pendant les tests d’oxygénation a été théoriquement évalué compte tenu des conditions d’opération. Par lasuite, des corrélations empiriques ont été construites en utilisant des nombres adimensionnels et expriment le coefficient kLa en fonction de la vitesse superficielle de gaz et la viscosité apparente, tout en considérant sa dépendance du taux de cisaillement. Enfin, le facteur alpha est défini comme une loi de puissance décroissante en fonction de la viscosité apparente, pour des systèmes à faible chargé. / The main purpose of this study was to evaluate and better understand the impact of superficial gas velocity and activated sludge properties,on activated sludge rheology and oxygen transfer in bioreactors. First of all, activated sludge rheology was evaluated using a tubular rheometer, designed and constructed in this work. Rheological measurements were performed with activated sludge from five different wastewater treatment plants and with MLSS concentrations between 2.3 and 10.2 g L-1. Results showed that although the sludge rheology is significantly defined by the MLSS concentration, other sludge characteristics related to the sludge orign, such as such as floc size, floc cohesiveness and floc density also influence the sludge apparent viscosity. Existing rheological models were evaluated on the set of obtained experimental flow curves. Besides, the oxygen transfer is evaluated in a bubble column (Hc=4.4, Dc=0.29 m) installed in two different wastewater treatment plants: a conventional activated sludge plant (CAS) and a membrane bioreactor (MBR). The column, alternatively equipped with a fine or a coarse bubble diffuser (FB, CB), was continuously fed with activated sludge extracted either from the aeration tank, the recirculation loop or the membrane reactor. With MLSS concentrations from 3.0 to 10.4 g L-1, the kkllaa coefficient was lower in activated sludge than in clean water and still reduced with an increase of the MLSS concentration. This reduction is partially attributed to the observed reduction of gas holdup (εεɢɢ), associated with an increase in the sludge apparent viscosity (µµαααααα), which leads to a reduction of the specific interfacial area (αα) due to bubble coalescence and the formation of larger bubbles. Besides, the concentration of non-ionic surfactants, exhibited a negative effect on the κκιιαα coefficient for the oxygenation tests performed under FB aeration conditions and low MLSS concentration. This impact was not observed under CB aeration conditions, which was explained by the higher renewal rates generated by coarse bubbles. Finally, the mean shear rate exerted by the bubble swarm in the column during the oxygen transfer tests was theoretically evaluated considering the operating conditions. Subsequently, empirical correlations were constructed using dimensionless numbers and express the oxygen transfer coefficient as a function of the superficial gas velocity and the apparent viscosity, considering its shear rate dependence. Finally, alpha factor is defined as a power law decreasing function of the apparent viscosity, for low loaded activated sludge systems.

Dissolved oxygen refers to the mass of oxygen that is contained in water. The concentration of dissolved oxygen is an important indicator of the environments water quality. The presence of oxygen in water is desirable therefore it is a positive sign; whereas the absence of oxygen is a sign of severe pollution. An adequate supply of dissolved oxygen is important for waste water treatment processes. Many naturally occurring biological and chemical processes use oxygen, thereby diminishing the dissolved oxygen concentration in the water. The physical process of oxygen transfer or oxygen absorption from the atmosphere acts to replenish the used oxygen. This process has been termed aeration. Aeration is the primary requirement of the biological treatment of water and wastewater treatment. As reported in the literature, the aeration process consumes as much as 60-80% of total power requirements in wastewater treatment plants. Therefore, it is necessary that the design and operation of aeration process should be economized in terms of their energy efficiency. The performance of surface aeration systems is rated in terms of their oxygen transfer rate; hence the choice of a particular surface aeration system depends on its performance and efficiency of oxygen transfer rates. Oxygen transfer rate and the corresponding power requirement to rotate the rotor are very vital parameters for the design and scale-up of surface aerators. Basically two types of operation are in use for surface aeration systems – batch operation and continuous operation. Batch operation involves a single vessel which is filled, aerated then completely emptied. Continuous operation method of operating a biological treatment plant is characterized by a steady input stream (in terms of chemical and biological composition and flow rate, when flow and concentration equalization is practiced), steady process conditions during the treatment steps and by a fairly consistent flow of treated material with only little variation in its composition. The work presented in this thesis consists of two parts. The first one deals with the experimental investigations on the three types of batch surface aeration tanks. A comprehensive design analysis has been worked out and presented on these types of surface aeration systems. In the second category, experimental investigations have been carried out extensively on continuous flow surface aeration systems of different sizes. Analysis has led to the formulation of optimal geometric dimension and the simulation criteria for the design purposes. As far as the first category of investigations is concerned, a substantial work has been reported on batch surface aerators on various issues, during the past several years. Still, a general methodology to scale up or scale down the process phenomena is lacking. In the present work, experiments were done on different shaped batch surface aeration system for generalizing or devising the scale up and scale down criteria for oxygen transfer coefficient and power consumption. Present work through experimental observations established that unbaffled circular tanks are more energy efficient than baffled when used as surface aerator. Power consumption in surface aeration systems is characterized by a fundamental non-dimensional parameter named power number. This number relates drag force to the inertial force in fluid flow system. Power number scaling up of unbaffled surface aerators of square, circular and rectangular shaped tanks is one of the most important contributions of the present work. Design charts have been developed for all the three shape of tanks for the installation as the batch surface aeration systems. Based on the experimental analyses in the present work, it was found that circular shape is the most efficient than any other shape and it is also established that generally a number of smaller sized tanks were more economic and efficient than using a single big tank while aerating the same volume of water. Based on the energy economy analysis, present work suggests the optimal speed range of batch systems of different shaped surface aeration tanks. Different sized rectangular aeration tanks with different aspect ratios (that is length to width ratio) were tested along with a series of square and circular tanks for comparing their relative performances. Present work by doing experiments answered this fact and found that square tank (aspect ratio =1) was more efficient than any other aspect ratio rectangular tanks. Vortexes are inherently present in any type of unbaffled tanks. Present work analyzed the vortex behavior of unbaffled surface aeration systems to determine the critical impeller speed in unbaffled batch surface aeration systems at which oxygen transfer rates are more. The second part of the present work establishes the optimal geometrical parameters of a continuous flow surface aeration systems. These types of operations were found to be least reported in the available literature and there appears to be of no report in the literature on optimal geometrical parameters. Extensive experimental work is reported in the present thesis on the establishment of the optimal geometrical parameters of continuous flow surface aeration systems. From there, simulation criteria are established by maintaining optimal geometrical similarity in different sized continuous flow surface aeration tanks; so that the scale up or scale down criteria can be applied to predict oxygen transfer rates and power number.

O ácido ƴ-poliglutâmico (ƴ-PGA) é uma homopoliamida aniônica, biodegradável, comestível e atóxica, sintetizada por bactérias do gênero Bacillus, podendo ser utilizado nas indústrias alimentícia e de cosméticos, na medicina e no tratamento de águas residuais. Este trabalho teve como objetivo caracterizar e identificar potenciais aplicações para o ƴ-PGA obtido através do cultivo submerso de Bacillus subtilis BL53, conduzido sob condições otimizadas em trabalhos anteriores. Além disso, foi avaliado o efeito de diferentes inóculos e da adição de precursores da rota metabólica na produção do biopolímero. A melhor condição obtida foi testada em biorreatores com adição de polidimetilsiloxano (PDMS) como carreador de oxigênio, com o objetivo de aumentar a produtividade do biopolímrero. A massa molar média (Mw), obtida através de espalhamento de luz estático, na ordem de 106 g mol-1 não apresentou diferenças significativas para o biopolímero obtido após 48 e 96 h de cultivo. As análises reológicas conduzidas em viscosímetro rotacional indicaram que os polímeros obtidos após 48 e 96 horas apresentaram comportamento Newtoniano, sendo que após 96 horas a viscosidade absoluta foi maior. As análises térmicas (calorimetria diferencial exploratória e análise termogravimétrica) indicaram a temperatura de fusão (Tm) de 134 ºC e 128 ºC e o intervalo de degradação (Td) entre 120 ºC - 190 ºC e 120 ºC - 215 ºC, para os biopolímeros obtidos após 48 e 96 horas de cultivo respectivamente. O caldo LB apresentou-se como o melhor inóculo para a produção de ƴ-PGA. A adição dos precursores L-glutamina e ácido -cetoglutárico aumentou em 20 % a produção do biopolímero. A adição de 10 % de PDMS nos cultivos em biorreatores aumentou o coeficiente volumétrico de transferência de massa (KLa) e a produção e produtividade do ƴ-PGA, sendo produzidos 23.5 g L-1 do biopolímero em 24 horas de cultivo, uma produtividade aproximadamente 40 % superior às obtidas por outros autores utilizando o mesmo microrganismo. / Poly-ƴ-glutamicacid (ƴ-PGA) is an anionic, biodegradable, non-toxic and edible homopolyamide, synthesized by bacteria of the genus Bacillus, being used in food, cosmetics, medicine and waste water treatment. The aim of this study is to characterize and indentify potencial applicatiions for the ƴ-PGA obtained by submerged cultivation of Bacillus subtilis BL53, conducted under optimized conditions in previous studies. We also evaluated the effect of different inoculants and addition of precursors in the metabolic pathway of production of the biopolymer. The best condition obtained yet been tested in bioreactors with addition of polydimethylsiloxane (PDMS) as a carrier of oxygen in order to further increase the productivity of biopolymer. The average molecular weight (Mw) obtained by static light scattering, on the order of 106 g mol-1, showed no significant differences for biopolymer obtained after 48 and 96 h of cultivation. Analyses conducted in rotational viscometer indicated that biopolymers after 48 and 96 h have a Newtonian behavior, and the 96 hours had higher absolute viscosity. The thermal analysis (differential scanning calorimetry and thermo gravimetric analysis) indicated the melting temperature (Tm) as 134 ºC and 128 ºC and degradation temperature range (Td) of 120 ºC - 190 ºC and 120 ºC - 215 ºC, after 48 and 96 hours respectively. It was found that the best inoculum medium for biopolymer production was the LB broth. The addition of the precursors L-glutamine and -ketoglutaric acid increased in 20% the ƴ-PGA production. The addition of 10% of PDMS in bioreactors cultures increased the mass transfer volumetric coefficient (KLa) and the production and productivity of ƴ-PGA, being produced 23.5 g l-1 of the biopolymer in 24 hours of cultivation, a productivity about 40 % higher than those obtained by other authors using the same microorganism.

O ácido y-poliglutâmico (y-PGA) é um biopolímero solúvel em água, aniônico, atóxico, biodegradável e biocompatível, produzido por Bacillus e que possui aplicações nas áreas química, médica, ambiental e de alimentos. Este trabalho tem como objetivo estudar o processo de produção do y-PGA a partir do cultivo de uma nova linhagem de Bacillus isolada de ambiente amazônico, o Bacillus subtilis BL53. Este estudo inclui a seleção de culturas, a otimização de condições de cultivo e a prospecção de substratos de produção alternativos. A habilidade de produção de y-PGA por linhagens de Bacillus isoladas na região amazônica foi investigada. A linhagem BL53 foi a linhagem selecionada, sendo identificada como Bacillus subtilis, através da análise da seqüência do gene 16S DNAr e de suas características bioquímicas. A avaliação das condições de cultivo submerso foi conduzida em agitador orbital mediante delineamento composto central rotacional (DCCR) que apontou como ponto ótimo de produção de y-PGA, a temperatura de 37 oC, o pH inicial de 6,9 e a concentração de 1,22 mM de Zn2+ suplementada ao Caldo E. Nas condições otimizadas, a produção de y-PGA foi igual a 10,4 g/L, cerca de três vezes maior que a obtida em condições convencionais de cultivo em Caldo E, utilizando a mesma linhagem. A influência da disponibilidade de oxigênio sobre a produção de y-PGA por B. subtilis BL53 foi avaliada em biorreator agitado de 5 L, com o emprego das velocidades de agitação de 500, 750 e 1000 rpm, sendo mantida fixa a taxa de aeração de 2 vvm. A produção de y-PGA mostrou-se altamente dependente da transferência de oxigênio, sendo que o teor de oxigênio dissolvido decaiu rapidamente nas primeiras 15 horas de cultivo, como resultado da elevada demanda de oxigênio pelas bactérias. O aumento na velocidade de agitação no biorreator possibilitou uma maior transferência de oxigênio e induziu o aumento na taxa de consumo de oxigênio pelas bactérias, conduzindo a maior produção e maior produtividade de y-PGA. A intensificação da agitação também influenciou os parâmetros cinéticos de crescimento do Bacillus subtilis BL53, provocando um aumento na velocidade específica de crescimento na fase logarítmica (max) sem provocar a perda da viabilidade celular. Com a utilização da velocidade de agitação de 1000 rpm em biorreator, o tempo de cultivo pode ser reduzido para menos de 48 h, cerca de 50% do tempo necessário para operação a 500 rpm. A investigação de substratos alternativos para produção de y-PGA mostrou o glicerol residual de biodiesel e o resíduo fibroso de soja como substratos promissores, apontando para a possibilidade de investigação em trabalhos futuros. / The poly-gamma-glutamic acid (y-PGA) is a water-soluble biopolymer, anionic, non toxic, biodegradable and biocompatible, it is produced by Bacillus and it has applications in chemical, medical, environmental and food industries. This work aims to study the process of production of y-PGA through cultivation of a new strain of Bacillus isolated from the Amazonian environment, Bacillus subtilis BL53. This study includes the screening of strains, the optimization of culture conditions and the investigation of alternative substrates. The ability of y-PGA production by Bacillus strains isolated from Amazonian environment was investigated. The BL53 strain was selected and identified as Bacillus subtilis, through analysis of 16S rDNA gene sequence and its biochemical characteristics. Evaluation of culture conditions in submerged cultivation was conducted in shaker using central composite design (CCD), which showed the temperature of 37 oC, the initial pH 6.9 and concentration of 1.22 mM Zn2+ in Medium E as the optimal conditions to y-PGA production. Under optimized conditions, the production of y-PGA was 10.4 g/L, about threefold what was obtained using this strain under conventional cultivation in Medium E. The influence of oxygen availability on the production of y-PGA by B. subitlis BL53 was evaluated in a 5 l stirred bioreactor with the use of stirring rates of 500, 750 and 1000 rpm, using a fixed aeration rate of 2 vvm. Production of y-PGA was highly dependent on oxygen transfer, and the dissolved oxygen content decreased rapidly in the first 15 hours of culture as a result of high oxygen demand by bacteria. The increase in stirring rate in the bioreactor allowed a better oxygen transfer and induced a rise in the oxygen uptake rate by bacteria, leading to higher production and higher productivity of y-PGA. The intensification of the stirring also influenced the kinetics growth parameters of Bacillus subtilis BL53, producing an increase in specific growth rate in the logarithmic phase (max) without causing reduction of cell viability. Using the stirring rate of 1000 rpm in a bioreactor, the cultivation time can be reduced to less than 48 h, about 50% of the time required for operation at 500 rpm. The investigation of alternative substrates for the production of y-PGA showed crude glycerol from biodiesel and soybean industrial fibrous residue as promising ones, pointing to research possibilities in future work.

O ácido ƴ-poliglutâmico (ƴ-PGA) é uma homopoliamida aniônica, biodegradável, comestível e atóxica, sintetizada por bactérias do gênero Bacillus, podendo ser utilizado nas indústrias alimentícia e de cosméticos, na medicina e no tratamento de águas residuais. Este trabalho teve como objetivo caracterizar e identificar potenciais aplicações para o ƴ-PGA obtido através do cultivo submerso de Bacillus subtilis BL53, conduzido sob condições otimizadas em trabalhos anteriores. Além disso, foi avaliado o efeito de diferentes inóculos e da adição de precursores da rota metabólica na produção do biopolímero. A melhor condição obtida foi testada em biorreatores com adição de polidimetilsiloxano (PDMS) como carreador de oxigênio, com o objetivo de aumentar a produtividade do biopolímrero. A massa molar média (Mw), obtida através de espalhamento de luz estático, na ordem de 106 g mol-1 não apresentou diferenças significativas para o biopolímero obtido após 48 e 96 h de cultivo. As análises reológicas conduzidas em viscosímetro rotacional indicaram que os polímeros obtidos após 48 e 96 horas apresentaram comportamento Newtoniano, sendo que após 96 horas a viscosidade absoluta foi maior. As análises térmicas (calorimetria diferencial exploratória e análise termogravimétrica) indicaram a temperatura de fusão (Tm) de 134 ºC e 128 ºC e o intervalo de degradação (Td) entre 120 ºC - 190 ºC e 120 ºC - 215 ºC, para os biopolímeros obtidos após 48 e 96 horas de cultivo respectivamente. O caldo LB apresentou-se como o melhor inóculo para a produção de ƴ-PGA. A adição dos precursores L-glutamina e ácido -cetoglutárico aumentou em 20 % a produção do biopolímero. A adição de 10 % de PDMS nos cultivos em biorreatores aumentou o coeficiente volumétrico de transferência de massa (KLa) e a produção e produtividade do ƴ-PGA, sendo produzidos 23.5 g L-1 do biopolímero em 24 horas de cultivo, uma produtividade aproximadamente 40 % superior às obtidas por outros autores utilizando o mesmo microrganismo. / Poly-ƴ-glutamicacid (ƴ-PGA) is an anionic, biodegradable, non-toxic and edible homopolyamide, synthesized by bacteria of the genus Bacillus, being used in food, cosmetics, medicine and waste water treatment. The aim of this study is to characterize and indentify potencial applicatiions for the ƴ-PGA obtained by submerged cultivation of Bacillus subtilis BL53, conducted under optimized conditions in previous studies. We also evaluated the effect of different inoculants and addition of precursors in the metabolic pathway of production of the biopolymer. The best condition obtained yet been tested in bioreactors with addition of polydimethylsiloxane (PDMS) as a carrier of oxygen in order to further increase the productivity of biopolymer. The average molecular weight (Mw) obtained by static light scattering, on the order of 106 g mol-1, showed no significant differences for biopolymer obtained after 48 and 96 h of cultivation. Analyses conducted in rotational viscometer indicated that biopolymers after 48 and 96 h have a Newtonian behavior, and the 96 hours had higher absolute viscosity. The thermal analysis (differential scanning calorimetry and thermo gravimetric analysis) indicated the melting temperature (Tm) as 134 ºC and 128 ºC and degradation temperature range (Td) of 120 ºC - 190 ºC and 120 ºC - 215 ºC, after 48 and 96 hours respectively. It was found that the best inoculum medium for biopolymer production was the LB broth. The addition of the precursors L-glutamine and -ketoglutaric acid increased in 20% the ƴ-PGA production. The addition of 10% of PDMS in bioreactors cultures increased the mass transfer volumetric coefficient (KLa) and the production and productivity of ƴ-PGA, being produced 23.5 g l-1 of the biopolymer in 24 hours of cultivation, a productivity about 40 % higher than those obtained by other authors using the same microorganism.

Made available in DSpace on 2016-06-02T19:56:54Z (GMT). No. of bitstreams: 1 5933.pdf: 2457795 bytes, checksum: b5b6653a532bc7e6c4a9ec2685b55277 (MD5) Previous issue date: 2014-08-15 / Universidade Federal de Minas Gerais / Owing to the broad biochemical, physiological and genetic characterization and the ease of manipulation and cultivation, several proteins with therapeutic and industrial applications are produced by genetically modified Escherichia coli. There is abundant information in the literature about strategies to increase biomass and recombinant protein production by cultivation of E. coli employing stirred and aerated tank bioreactor (conventional type). In contrast, although quite employed in cultivation of filamentous microorganisms, there are just a few studies involving cultivation of E. coli in airlift bioreactor. This reactor offers advantages over conventional one, such as simplicity of construction, reduced risk of contamination and efficient gas-liquid dispersion with low power consumption. However, the lower O2 transfer in airlift bioreactor (bench scale), compared to that achieved in conventional one, justifies the manipulation of temperature, pressure and flow rates of air and O2 in the dissolved oxygen concentration (DOC) control, avoiding decreases in biomass and recombinant protein productivities. In this context, this study aimed to: (i) determine the influence of internal pressure on the gas holdup (ɛ), volumetric O2 transfer coefficient (kLa) and volumetric O2 transfer rate (continue...). / Devido à ampla caracterização bioquímica, fisiológica e genética e à facilidade de manipulação e de cultivo, diversas proteínas com aplicação industrial e terapêutica são produzidas por Escherichia coli geneticamente modificada. Há informações abundantes na literatura sobre estratégias para aumentar a produção de biomassa e de proteínas recombinantes por meio de cultivos de E. coli empregando biorreator tipo tanque agitado e aerado (convencional). Por outro lado, apesar de bastante empregado em cultivos de microrganismos filamentosos, poucos trabalhos envolvendo cultivo de E. coli em reator pneumático tipo airlift são encontrados. Este biorreator apresenta vantagens frente ao convencional, como simplicidade de construção, menor risco de contaminação e eficiente dispersão gás-líquido com baixo consumo de energia. Entretanto, a menor capacidade de transferência de O2 no biorreator airlift em escala de bancada, em relação à alcançada em reator convencional, justifica a manipulação de variáveis como temperatura, pressão e vazões de ar e O2 no controle adequado da concentração de O2 dissolvido (COD), evitando quedas de produtividade de biomassa e de proteína recombinante. Nesse contexto, este trabalho objetivou: (i) determinar a influência da pressão interna sobre a retenção gasosa (ɛ), o coeficiente volumétrico de transferência de O2 (kLa) e a velocidade volumétrica de transferência de O2 (continua...).

O ácido y-poliglutâmico (y-PGA) é um biopolímero solúvel em água, aniônico, atóxico, biodegradável e biocompatível, produzido por Bacillus e que possui aplicações nas áreas química, médica, ambiental e de alimentos. Este trabalho tem como objetivo estudar o processo de produção do y-PGA a partir do cultivo de uma nova linhagem de Bacillus isolada de ambiente amazônico, o Bacillus subtilis BL53. Este estudo inclui a seleção de culturas, a otimização de condições de cultivo e a prospecção de substratos de produção alternativos. A habilidade de produção de y-PGA por linhagens de Bacillus isoladas na região amazônica foi investigada. A linhagem BL53 foi a linhagem selecionada, sendo identificada como Bacillus subtilis, através da análise da seqüência do gene 16S DNAr e de suas características bioquímicas. A avaliação das condições de cultivo submerso foi conduzida em agitador orbital mediante delineamento composto central rotacional (DCCR) que apontou como ponto ótimo de produção de y-PGA, a temperatura de 37 oC, o pH inicial de 6,9 e a concentração de 1,22 mM de Zn2+ suplementada ao Caldo E. Nas condições otimizadas, a produção de y-PGA foi igual a 10,4 g/L, cerca de três vezes maior que a obtida em condições convencionais de cultivo em Caldo E, utilizando a mesma linhagem. A influência da disponibilidade de oxigênio sobre a produção de y-PGA por B. subtilis BL53 foi avaliada em biorreator agitado de 5 L, com o emprego das velocidades de agitação de 500, 750 e 1000 rpm, sendo mantida fixa a taxa de aeração de 2 vvm. A produção de y-PGA mostrou-se altamente dependente da transferência de oxigênio, sendo que o teor de oxigênio dissolvido decaiu rapidamente nas primeiras 15 horas de cultivo, como resultado da elevada demanda de oxigênio pelas bactérias. O aumento na velocidade de agitação no biorreator possibilitou uma maior transferência de oxigênio e induziu o aumento na taxa de consumo de oxigênio pelas bactérias, conduzindo a maior produção e maior produtividade de y-PGA. A intensificação da agitação também influenciou os parâmetros cinéticos de crescimento do Bacillus subtilis BL53, provocando um aumento na velocidade específica de crescimento na fase logarítmica (max) sem provocar a perda da viabilidade celular. Com a utilização da velocidade de agitação de 1000 rpm em biorreator, o tempo de cultivo pode ser reduzido para menos de 48 h, cerca de 50% do tempo necessário para operação a 500 rpm. A investigação de substratos alternativos para produção de y-PGA mostrou o glicerol residual de biodiesel e o resíduo fibroso de soja como substratos promissores, apontando para a possibilidade de investigação em trabalhos futuros. / The poly-gamma-glutamic acid (y-PGA) is a water-soluble biopolymer, anionic, non toxic, biodegradable and biocompatible, it is produced by Bacillus and it has applications in chemical, medical, environmental and food industries. This work aims to study the process of production of y-PGA through cultivation of a new strain of Bacillus isolated from the Amazonian environment, Bacillus subtilis BL53. This study includes the screening of strains, the optimization of culture conditions and the investigation of alternative substrates. The ability of y-PGA production by Bacillus strains isolated from Amazonian environment was investigated. The BL53 strain was selected and identified as Bacillus subtilis, through analysis of 16S rDNA gene sequence and its biochemical characteristics. Evaluation of culture conditions in submerged cultivation was conducted in shaker using central composite design (CCD), which showed the temperature of 37 oC, the initial pH 6.9 and concentration of 1.22 mM Zn2+ in Medium E as the optimal conditions to y-PGA production. Under optimized conditions, the production of y-PGA was 10.4 g/L, about threefold what was obtained using this strain under conventional cultivation in Medium E. The influence of oxygen availability on the production of y-PGA by B. subitlis BL53 was evaluated in a 5 l stirred bioreactor with the use of stirring rates of 500, 750 and 1000 rpm, using a fixed aeration rate of 2 vvm. Production of y-PGA was highly dependent on oxygen transfer, and the dissolved oxygen content decreased rapidly in the first 15 hours of culture as a result of high oxygen demand by bacteria. The increase in stirring rate in the bioreactor allowed a better oxygen transfer and induced a rise in the oxygen uptake rate by bacteria, leading to higher production and higher productivity of y-PGA. The intensification of the stirring also influenced the kinetics growth parameters of Bacillus subtilis BL53, producing an increase in specific growth rate in the logarithmic phase (max) without causing reduction of cell viability. Using the stirring rate of 1000 rpm in a bioreactor, the cultivation time can be reduced to less than 48 h, about 50% of the time required for operation at 500 rpm. The investigation of alternative substrates for the production of y-PGA showed crude glycerol from biodiesel and soybean industrial fibrous residue as promising ones, pointing to research possibilities in future work.

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No. of bitstreams: 1 TeseGYR.pdf: 8156092 bytes, checksum: 727d9cd338ae7ed90fe24238b42228bd (MD5) Previous issue date: 2015-03-25 / Outra / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Pneumatic bioreactors are devices free of moving parts which have the purpose of converting raw materials in bio-products of commercial interest by the action of enzymes or cells. They are promising in the biochemical industry, ensuring good oxygen transfer and consuming less energy. Global performance parameters such as global gas hold up and the volumetric oxygen transfer coefficient are important criteria in the design and selection among different geometries of the mentioned devices. In the present work it was carried out modeling and simulation of pneumatic bioreactors based on Computational Fluid Dynamics (CFD) in order to estimate the global gas hold up and the volumetric oxygen transfer coefficient in three different geometries of pneumatic bioreactors: bubble column, concentric tube airlift and split tube airlift. The simulated results of each performance parameter were verified by comparison with the experimental values reported by Thomasi et al. (2010) and Mendes and Badino (2015) for the fluids distilled water, glycerol solution 10 cP and xanthan gum solution 0.2% w/v (weight/volume) in a wide range of specific air flow rate (0 to 5 min- 1). Application suite ANSYS® 14.5 was used for numerical simulations in CFD. Important parameters such as the bubble diameter played a great influence on results of the volumetric oxygen transfer coefficient. It can be observed by the experimental and simulated results that the concentric tube airlift bioreactor was the best alternative to the global gas hold up and the volumetric oxygen transfer coefficient (reaching 14% and 0.06 s-1 for distilled water, respectively). It was found that the results obtained via the CFD agreed with the majority trend of experimental data, capturing the most important hydrodynamic phenomena and mass transfer characteristics, showing that the modeling of different systems with different fluids fulfilled the main objective of obtaining reliable models design and performance of other geometries of pneumatic bioreactors. / Os biorreatores pneumáticos são equipamentos industriais isentos de partes móveis que têm a finalidade de converter matérias-primas em bioprodutos de interesse comercial pela ação de enzimas ou células. São promissores na indústria bioquímica, pois garantem boa transferência de oxigênio consumindo menos energia. Parâmetros globais de desempenho, como a retenção gasosa global e o coeficiente volumétrico de transferência de oxigênio são critérios importantes no projeto e seleção entre geometrias diferentes dos equipamentos mencionados. No presente trabalho foi realizada a modelagem e a simulação de biorreatores pneumáticos baseada na Fluidodinâmica Computacional (CFD - Computational Fluid Dynamics) de forma a estimar a retenção gasosa global e o coeficiente volumétrico de transferência de oxigênio em três geometrias distintas: coluna de bolhas, airlift de cilindros concêntricos e airlift split. Os resultados simulados de cada parâmetro de desempenho foram verificados comparando-se com os valores experimentais reportados nos trabalhos de Thomasi et al. (2010) e Mendes e Badino (2015) para os fluidos água destilada, solução de glicerol 10 cP e solução de goma xantana 0,2% m/v (massa/volume) e vazão de alimentação específica de ar numa ampla faixa (0 a 5 min-1). Foi empregada a suíte de aplicativos ANSYS® 14.5 para as simulações numéricas em CFD. Parâmetros importantes, como o diâmetro de bolha, exerceram grande influência nos resultados referentes ao coeficiente volumétrico de transferência de oxigênio. Destaca-se, pelos resultados experimentais e simulados, que o biorreator pneumático do tipo airlift de cilindros concêntricos apresentou-se como a melhor alternativa para a retenção gasosa global e para o coeficiente volumétrico de transferência de oxigênio (atingindo 14% e 0,06 s-1 para a água destilada, respectivamente). Verificou-se que os resultados obtidos via CFD concordaram com a tendência majoritária dos dados experimentais, capturando os fenômenos mais relevantes das características hidrodinâmicas e da transferência de massa, mostrando que a modelagem dos diferentes sistemas com diferentes fluidos atendeu ao principal objetivo de obter modelos confiáveis para o projeto e comparação de desempenho de outras geometrias de biorreatores pneumáticos. / CNPq: 478472/2011-0 / CNPq: 140466/2011-8