A transferência de oxigênio é um fator limitante para grande parte dos cultivos em biorreatores que operam com organismos estritamente aeróbios devido à baixa solubilidade do oxigênio em meios de cultivo. A introdução de meios não convencionais, como polidimetilsiloxanos (PDMS), em biorreatores pode ser vista como uma melhoria para o ajuste desses processos. Este trabalho propõe uma investigação do emprego de polidimetilsiloxano fluido e emulsificado, usados como carreadores de oxigênio, em meios de cultivo na produção de lipases de Staphylococcus warneri EX17, cepa capaz de utilizar glicerol residual como substrato, visando melhorar a disponibilidade de oxigênio e aperfeiçoar a produtividade. Inicialmente, foram realizados estudos para selecionar o uso de glicerol residual, oriundo da síntese química de biodiesel como fonte de carbono. A atividade lipolítica foi semelhante com glicerol comercial e residual. Após a seleção do glicerol foram realizados experimentos em incubadora rotatória horizontal nas condições previamente otimizadas em trabalhos anteriores do grupo, adicionando PDMS. Dois tipos de PDMS foram testados, fluido e emulsificado. A produção de lipase foi significativamente maior no meio contendo PDMS. Assim, ferramentas de planejamento experimental, delineamento composto central (DCC) foram utilizadas para verificar a influencia do PDMS no coeficiente de transferência de oxigênio kLa em meio de cultivo produtor de lipase livre de células. O kLa aumentou significativamente no meio de cultivo contendo PDMS. Dois novos planejamentos foram desenhados, para otimizar a produção de lipase em meio de cultivo com células. Um para PDMS fluido e outro para PDMS emulsificado. No meio contendo PDMS fluido, a atividade lipolítica foi cinco vezes maior, enquanto que no meio contendo silicone emulsificado a atividade lipolítica foi três vezes maior. Este estudo demonstrou que a lipase de S. warneri EX17 pode ser produzida utilizando glicerol residual como fonte de carbono, e polidimetilsiloxanos como carreadores de oxigênio aumentando a transferência de oxigênio no meio e elevando a produção da enzima, que apresenta diversas possibilidades de aplicação, principalmente na indústria de alimentos. / The oxygen transfer is a limiting factor in many bioreactors cultivations, in which strictly aerobic organisms are grown due to the low solubility of oxygen in culture media. The introduction of unconventional media, such as polydimethylsiloxanes (PDMS) in bioreactors can be seen as a potential improvement for these processes. This work proposes an investigation of the use of polydimethylsiloxane in its two forms, fluid and emulsified, used as carriers of oxygen in culture media for the lipase production of Staphylococcus warneri EX17, strain capable of using glycerol as substrate, to improve the oxygen availability and improve enzyme productivity. Initially, studies were performed to select the use of residual glycerol, derived from the chemical synthesis of biodiesel as a source of carbon. The lipase activity was similar in both commercial and residual glycerol. Experiments were performed on orbital shaker, using conditions previously optimized in another work of the group, but with the addition of PDMS. Two types of PDMS were tested, the fluid and the emulsified. The lipase production was significantly higher in medium containing PDMS. Experimental design and central composite design (CCD) were used to evaluate the influence of PDMS on the oxygen transfer coefficient kLa in culture medium free of lipase-producing cells. The kLa increased significantly in the medium containing PDMS. Two new experimental plannings were designed to optimize the production of lipase in culture medium with cells. In the medium containing the PDMS fluid lipase activity was five times higher than medium without the oxygen carrier, while in the medium containing the emulsified silicone lipase activity was three times higher. This study showed that the lipase from S. warneri EX17 can be produced using residual glycerol as carbon source and that polydimethylsiloxanes works as interesting carrier of oxygen by increasing oxygen transfer rates, improving enzyme production, which has several possible applications, especially in the food industry.

Orientador: Marcel Otavio Cerri / Resumo: Streptomyces clavuligerus é uma bactéria filamentosa, capaz de produzir ácido clavulânico (AC), um importante fármaco β-lactâmico. Sendo necessário o controle de parâmetros como pH, temperatura, homogeneização do caldo, adequada transferência de oxigênio e cisalhamento para melhora de processos biotecnológicos. Sabe-se que a partir da transferência de oxigênio e da velocidade média de cisalhamento imposta ao sistema nesses biorreatores, por meio do uso de maiores ou menores rotações dos impelidores, a produção de ácido clavulânico é afetada, pois essas variáveis influenciam no crescimento do microrganismo, na reologia e morfologia do microrganismo presente no caldo fermentativo e na formação e estabilidade da molécula de AC. No presente trabalho, foram realizados experimentos para determinação volumétrica de oxigênio com diferentes conformações para encontrar o melhor posicionamento dos impelidores. Com a melhor conformação, estudou- se a transferência de oxigênio com diferentes vazões e rotações de ar. Os resultados desses experimentos mostram que um sistema pode apresentar melhores ou piores resultados de transferência de oxigênio para o processo, dependendo da sua geometria. A variação na distância entre os impelidores aumentou em até 83% a transferência de oxigênio do biorreator. Foram realizados cultivos de Streptomyces clavuligerus em biorreator convencional com diferentes vazões, rotações e composição de gás para comparar os diversos efeitos sofridos na morfologia dos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Streptomyces clavuligerus is a filamentous bacterium, producing of clavulanic acid (CA), an important β-lactam drug. It is necessary to control parameters such as pH, temperature, broth homogenization, adequate oxygen transfer and shear to improve biotechnological processes. Knows that from the transfer of oxygen and the average shear velocity imposed to the system in these bioreactors, through the use of greater or smaller rotations of the impellers, affect the production of clavulanic acid, since these variables influence the growth of the microorganism, in the rheology and morphology of the microorganism present in the fermentative broth and in the formation and stability of the CA molecule. In the present work, experiments were carried out for the volumetric determination of oxygen with different conformations to find the best positioning of the impellers. With the best conformation, the transfer of oxygen with different flow rates and air rotations was studied. The results of these experiments show that a system can present better or worse oxygen transfer results to the process depending on its geometry. The variation in distance between the impellers increased by up to 83% the transfer of oxygen from the bioreactor. Streptomyces clavuligerus cultures were grown in conventional bioreactor with different flow rates, rotations and gas composition to compare the different effects of microorganism’s morphology, substrate consumption, optical density and rheology and clavulan... (Complete abstract click electronic access below) / Mestre

<p>This master thesis in energy optimization was made during the autumn of 2006 at Käppala wastewater treatment plant in Lidingö, Stockholm. A preceding thesis, where all electricity consumption was mapped, showed that the aeration in the biological treatment is the single largest consumer in the plant, and it is therefore of interest to reduce this cost. The oxygen control strategy used at Käppala WWTP is working well from a nutrient removal point of view, but not from an economic one. The last aerobic zones have a very low oxygen consumption during low loading periods which give rise to enhanced dissolved oxygen concentrations with excessive costs and reduced denitrification as a result. But also during periods of normal loading unnecessary high oxygen concentration are sometimes given.</p><p>By modifying the aeration control strategy three full-scale experiments have been made, with the intention to reduce the air consumption. The experiments were carried out during week 37-50 in the autumn of 2006 and showed that savings could be made.</p><p>The regular oxygen control at Käppala WWTP controls the oxygen level in the aerobic compartment with two DO-setpoints; one in the first aerobic zone and one in the last. The zones in between are controlled by an airflow fractionation depending on the oxygen level in the first and last zone. In the first strategy to be evaluated, all four zones in the aerated part were individually controlled with its own setpoint. Two different setpoint combinations were tested. By using the fact that the efficiency in the oxygen transfer rate was higher at low airflows, savings of approximately 16 % were achieved. In the second strategy tested, an ammonia-feedback control combined with a DO-feedback controlled the DO-set point in the first aerobic zone. This strategy adjusted the DO- set points to the loading variations, and this gave a decreased airflow of approximately 9 %. Finally the two strategies were combined. All zones were then controlled individually with DO-set points set by an ammonium-feedback and a DO-feedback. The strategy gave savings in the airflow of approximately 18 %. In all three trials the aerated zones were more efficiently used, and the estimated savings are 550 000 SEK/year, and with a preserved nutrient removal efficiency.</p> / <p>Under hösten 2006 har ett examensarbete om energieffektivisering på Käppalaverket på Lidingö utförts. Ett föregående examensarbete där all elenergiförbrukning kartlades visade att blåsmaskinerna i biosteget står för den enskilt största förbrukningen i verket och det är därför av intresse att minska denna kostnad. Syrestyrningsstrategin som används på Käppalaverket fungerar mycket bra ur reningssynpunkt, men är inte optimal ur energisynpunkt. Dels luftas de första aeroba zonerna för mycket vid låg belastning vilket ger upphov till kraftigt förhöjda syrekoncentrationer i de sista aeroba zonerna med höga luftningskostnader och risk för försämrad denitrifikation, men även under normal belastning har det visat sig att onödigt höga syrekoncentrationer ibland ges.</p><p>Tre fullskaliga optimeringsförsök har utförts, med syfte att minska luftförbrukningen med bibehållen reningsgrad. Försöken pågick från vecka 37 till 50 hösten 2006, och visade att det finns möjlighet att spara energi genom att modifiera syrestyrningsstrategin.</p><p>Den reguljära syreregleringen i Käppalaverket styr syrehalten i den aeroba bassängen mot två syrebörvärden; ett i den första luftade zonen och ett i den sista. Luftflödet till de mellanliggande zonerna styrs av luftflödesandelar beroende på syrehalten i dessa två zoner. Den första strategin som utvärderades styrde istället samtliga zoner individuellt med egna börvärden, där två olika strukturer på de satta börvärdena användes. Genom att utnyttja en högre effektivitet i syreöverföringshastigheten vid låga luftflöden uppnåddes luftflödesbesparingar på ca 16 % i första försöket.</p><p>I den andra strategin styrdes syrebörvärdet i den första luftade zonen med hjälp av två återkopplingar, en från utgående ammoniumhalt och en från syrehalten i den sista luftade zonen.</p><p>Tack vare att strategin anpassade syrebörvärdena efter belastningen av syretärande ämnen erhölls luftflödesbesparingar på ca 9 %. Slutligen kombinerades de två strategierna; samtliga zoner styrdes individuellt med börvärden satta av en ammonium-återkoppling och en syre-återkoppling. Strategin medförde luftflödesbesparingar på ca 18 %. I samtliga försök utnyttjades de luftade zonerna bättre, och besparingspotentialen uträknad från 2005 års elpriser blev som mest 550 000 SEK/år, detta med en bibehållen reningsgrad.</p>

This master thesis in energy optimization was made during the autumn of 2006 at Käppala wastewater treatment plant in Lidingö, Stockholm. A preceding thesis, where all electricity consumption was mapped, showed that the aeration in the biological treatment is the single largest consumer in the plant, and it is therefore of interest to reduce this cost. The oxygen control strategy used at Käppala WWTP is working well from a nutrient removal point of view, but not from an economic one. The last aerobic zones have a very low oxygen consumption during low loading periods which give rise to enhanced dissolved oxygen concentrations with excessive costs and reduced denitrification as a result. But also during periods of normal loading unnecessary high oxygen concentration are sometimes given. By modifying the aeration control strategy three full-scale experiments have been made, with the intention to reduce the air consumption. The experiments were carried out during week 37-50 in the autumn of 2006 and showed that savings could be made. The regular oxygen control at Käppala WWTP controls the oxygen level in the aerobic compartment with two DO-setpoints; one in the first aerobic zone and one in the last. The zones in between are controlled by an airflow fractionation depending on the oxygen level in the first and last zone. In the first strategy to be evaluated, all four zones in the aerated part were individually controlled with its own setpoint. Two different setpoint combinations were tested. By using the fact that the efficiency in the oxygen transfer rate was higher at low airflows, savings of approximately 16 % were achieved. In the second strategy tested, an ammonia-feedback control combined with a DO-feedback controlled the DO-set point in the first aerobic zone. This strategy adjusted the DO- set points to the loading variations, and this gave a decreased airflow of approximately 9 %. Finally the two strategies were combined. All zones were then controlled individually with DO-set points set by an ammonium-feedback and a DO-feedback. The strategy gave savings in the airflow of approximately 18 %. In all three trials the aerated zones were more efficiently used, and the estimated savings are 550 000 SEK/year, and with a preserved nutrient removal efficiency. / Under hösten 2006 har ett examensarbete om energieffektivisering på Käppalaverket på Lidingö utförts. Ett föregående examensarbete där all elenergiförbrukning kartlades visade att blåsmaskinerna i biosteget står för den enskilt största förbrukningen i verket och det är därför av intresse att minska denna kostnad. Syrestyrningsstrategin som används på Käppalaverket fungerar mycket bra ur reningssynpunkt, men är inte optimal ur energisynpunkt. Dels luftas de första aeroba zonerna för mycket vid låg belastning vilket ger upphov till kraftigt förhöjda syrekoncentrationer i de sista aeroba zonerna med höga luftningskostnader och risk för försämrad denitrifikation, men även under normal belastning har det visat sig att onödigt höga syrekoncentrationer ibland ges. Tre fullskaliga optimeringsförsök har utförts, med syfte att minska luftförbrukningen med bibehållen reningsgrad. Försöken pågick från vecka 37 till 50 hösten 2006, och visade att det finns möjlighet att spara energi genom att modifiera syrestyrningsstrategin. Den reguljära syreregleringen i Käppalaverket styr syrehalten i den aeroba bassängen mot två syrebörvärden; ett i den första luftade zonen och ett i den sista. Luftflödet till de mellanliggande zonerna styrs av luftflödesandelar beroende på syrehalten i dessa två zoner. Den första strategin som utvärderades styrde istället samtliga zoner individuellt med egna börvärden, där två olika strukturer på de satta börvärdena användes. Genom att utnyttja en högre effektivitet i syreöverföringshastigheten vid låga luftflöden uppnåddes luftflödesbesparingar på ca 16 % i första försöket. I den andra strategin styrdes syrebörvärdet i den första luftade zonen med hjälp av två återkopplingar, en från utgående ammoniumhalt och en från syrehalten i den sista luftade zonen. Tack vare att strategin anpassade syrebörvärdena efter belastningen av syretärande ämnen erhölls luftflödesbesparingar på ca 9 %. Slutligen kombinerades de två strategierna; samtliga zoner styrdes individuellt med börvärden satta av en ammonium-återkoppling och en syre-återkoppling. Strategin medförde luftflödesbesparingar på ca 18 %. I samtliga försök utnyttjades de luftade zonerna bättre, och besparingspotentialen uträknad från 2005 års elpriser blev som mest 550 000 SEK/år, detta med en bibehållen reningsgrad.

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.

Parmi les méthodes alternatives respectueuses de l'environnement pour la lutte contre les ravageurs et la protection des cultures agricoles, les bioinsecticides à base de Bacillus thuringiensis sont les plus largement utilisés. L’activité insecticide de B.thuringiensis réside dans la production d’inclusions cristallines protéiques appelées δ-endotoxines qui sont formées au cours de la sporulation. En préalable à ces travaux, de nouvelles souches de B.thuringiensis ont été isolées du sol libanais et ont montré un potentiel insecticide élevé, supérieur à des souches classiquement utilisées. Dans le cadre du développement d'une production industrielle, au Liban, de biopesticides à base de ces souches de B.thuringiensis, ce travail a visé à optimiser la production des δ-endotoxines afin d'augmenter les rendements et de réduire les coûts de production. A l'échelle industrielle, le milieu de culture des bactéries représente une part importante du coût de production. Ainsi, l'effet, sur la concentration des δ-endotoxines, de substitution des sources synthétiques coûteuses d'azote, de carbone et de minéraux, par un sous-produit de la minoterie de blé (le son de blé) a été étudié. L’efficacité de ce sous-produit agro-industriel comme source complète de nutriments, disponible localement à faible prix, a été prouvée pour des fermentations submergées de deux souches différentes toxiques contre des diptères ou des lépidoptères. La comparaison du milieu son de blé à d’autres milieux (synthétiques ou non), classiquement utilisés en bibliographie, a montré que ce milieu présente de nombreux avantages en termes de productivité, de rendement relatif des δ-endotoxines et de coût. Parallèlement à ce travail, une méthode simple et rapide de purification des cristaux a été développée de façon à obtenir des cristaux protéiques purs à 99 % nécessaires comme standards pour le dosage des cristaux dans le milieu de fermentation. Un autre objectif du travail a été d’analyser l'effet de l'aération sur la cinétique de croissance et de sporulation d'une nouvelle souche de B.thuringiensis var kurstaki. Les différentes conditions d’aération testées en fermenteurs de 2 L, caractérisées par le coefficient volumétrique de transfert de dioxygène (KLa), ont permis de montrer l’importance de ce paramètre et de l’optimiser pour obtenir la meilleure productivité en biopesticides. Le meilleur rendement en protéines de toxines a été obtenu dans un milieu son de blé 6 % (masse/volume) dans des conditions d’aération correspondant à un KLa de 65,5 h-1 alors que la meilleure productivité des protéines de toxines a été atteinte dans le milieu 9 % pour un KLa de 102 h-1. Sur la base de cette étude, le dimensionnement de la production de B.thuringiensis à l’échelle 1000 L a été réalisé. L’unité de production a été construite et les premiers essais réalisés ont permis de produire de façon satisfaisante des lots de biopesticides. L’ajustement des protocoles industriels reste à faire. / Among the safe and environment friendly methods for the fight against pests and agricultural crops protection, Bacillus thuringiensis based biopesticides are the most widely used. The insecticidal activity of B. thuringiensis resides in the production of protein crystal inclusions called δ-endotoxins, which are formed during sporulation. Preliminary to this work, new B. thuringiensis strains were isolated from Lebanese soil and showed higher insecticidal potential than the conventionally used strains. As part of the development of an industrial production, in Lebanon, of biopesticides based on these strains of B.thuringiensis, this work aimed to optimize the production of δ-endotoxins to increase yields and reduce the cost of mass production. At industrial scale, the bacteria culture medium represents a significant part of the production cost. Thus, the effect on the concentration of the δ-endotoxins, of substituting expensive synthetic sources of nitrogen, carbon, and minerals, with a byproduct of wheat mill (wheat bran) was studied. The effectiveness of this agro-industrial by-product available locally at low prices, as complete source of nutrients, in submerged fermentations of two different strains of B.thuringiensis toxic against Lepidoptera and Diptera, has been proven. The comparison of the wheat bran medium to other mediums (synthetic or not), conventionally used in the bibliography, has shown that this medium this medium has many advantages in terms of productivity, relative yield of δ-endotoxins and cost. In parallel to this work, a simple and quick method of crystal purification was developed to obtain pure protein crystals to 99 % required as standards for the determination of crystals concentration in the fermentation medium. Another objective of the study was to analyze the effect of aeration on growth and sporulation kinetics of a new strain of B.thuringiensis var kurstaki. The different aeration conditions tested in fermenters of 2 L, characterized by the volumetric oxygen transfer coefficient (KLa), permitted to show the importance of this parameter and to optimize it to get the best biopesticides productivity. The best yield of toxin proteins relative to the wheat bran ratio in the culture medium was obtained in the 6 % (weight/volume) medium in aeration conditions corresponding to a KLa of 65.5 h-1, while the highest productivity of toxin proteins has been reached in the 9 % medium for KLa of 102 h-1. On this study basis, the scale up of B. thuringiensis production was performed in a bioreactor of 1000 L. The production unit was built and the first tests carried out have produced satisfactory batches of biopesticides. Industrial protocols are still to be adjusted.

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 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.

L'acide succinique est une molécule linéaire, bi-fonctionnelle qui possède de nombreuses applications alimentaires, chimiques et pharmaceutiques etc. Les connaissances de la régulation des voies métaboliques d’organismes d’intérêt industriel, le génie génétique et le génie des procédés ont permis à des microorganismes recombinants (C. glutamicum) de produire jusqu'à 100 g.L-1 de succinate avec des rendements intéressants. C. glutamicum est largement connu comme l'un des meilleurs producteurs industriels de nombreux acides aminés (glutamate, lysine etc.). Cependant, des études de C. glutamicum ont démontré sa capacité à produire plusieurs acides organiques (succinate, lactate, acétate, etc.). Au cours de ce travail, nous avons supprimé le gène ldhA de C. glutamicum en utilisant le plasmide pk19mobsacBΔldhA. Nous avons démontré que la délétion de ce gène n’avait pas d’incidence sur la capacité de croissance de la bactérie. Par ailleurs, nous avons étudié les effets de l’oxygénation sur la réponse physiologique de C. glutamicum 2262ΔldhA à travers des expériences de cultures en fioles en verre lisses en imposant différentes conditions de kLa. Les résultats ont montré que des faibles kLa (<33 h-1) favorisaient la production d’acides organiques tandis que les kLa élevés amélioraient surtout l’accumulation de la biomasse. Nous avons également mis en œuvre un procédé de production très efficace avec une phase de transition aérobiose-anaérobiose basée sur la régulation de la concentration en oxygène dissous. Avec ce procédé, 327 mM de succinate avec un rendement de 0,94 mole par mole de glucose ont pu être produits avec le mutant ΔldhA. En outre, nous avons vérifié l’efficacité de ce nouveau procédé en l’appliquant à la souche sauvage qui normalement produit 10 fois plus de lactate que de succinate. Ces résultats ont montré une production de 793 mM (94 g.L-1) de succinate et 785 mM (71 g.L-1) de lactate. Ils soulignent ainsi, l'importance de la phase de transition aérobiose-anaérobiose lors des procédés de production de succinate par des bactéries aérobie facultatif. Enfin, des expériences en système bi-étagé ont montré que C. glutamicum 2262 pouvait s’adapter très facilement aux gradients et hétérogénéités en oxygène dissous dans les cultures à grande échelle / Succinic acid is a linear and bi-functional molecule that has several practical applications including food, chemical and pharmaceutical industries. Thanks to increased knowledge on metabolism and pathway regulation of industrially relevant organisms, to the development of performant genetic tools and process engineering, recombinants strains (Escherichia coli, Corynebacterium glutamicum etc.) have been reported to be able to produce up to 100 g.L-1 with interesting yields (> 1.5 mole per mole glucose). C. glutamicum is well known as one of the best industrial producers of numerous amino acids (glutamate, lysine etc.). However, recent studies of C. glutamicum revealed its capability to produce several organic acids (succinate, lactate, acetate, etc.). In this work, we have deleted the ldhA gene of C. glutamicum by using a plasmid vector pk19mobsacBΔldhA. We demonstrated that the mutant and the wild type presented similar growth kinetics with maximal growth rate of about 0.7 h-1. We studied also the effects of oxygenation on C. glutamicum 2262 ΔldhA through cultures at different kLa and it appeared that lower kLa (<33 h-1) favored organic acids production wile higher favored bacterial growth. Furthermore, we designed a tri-phasic process with transition phase by regulation of dissolved oxygen concentration which resulted in the production of 327 mM of succinic acid with a yield of 0.94 mole per mole glucose. The application of the designed process to C. glutamicum 2262 wild type that normally produces lactate with a lactate to succinate production ratio up to 13.3 mol.mol-1, resulted in succinate concentration up to 793 mM (94 g.L-1) and 785 mM (71 g.L-1) of lactate. The succinate production yield was 1.1 mole per mole glucose and acetate production was negligible. These results underlined the importance of aerobic to anaerobic transition in succinate production processes of facultative aerobes and the necessity to engineer not only the microorganism but also the process. Finally, scale-down study have demonstrated the robustness of C. glutamicum against the oxygen gradients in bioreactor

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-05T19:18:20Z No. of bitstreams: 1 TeseCEMac.pdf: 12049463 bytes, checksum: acd65707762e60b912f35a2d267ad5b7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:40:54Z (GMT) No. of bitstreams: 1 TeseCEMac.pdf: 12049463 bytes, checksum: acd65707762e60b912f35a2d267ad5b7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:41:14Z (GMT) No. of bitstreams: 1 TeseCEMac.pdf: 12049463 bytes, checksum: acd65707762e60b912f35a2d267ad5b7 (MD5) / Made available in DSpace on 2016-10-20T13:41:32Z (GMT). No. of bitstreams: 1 TeseCEMac.pdf: 12049463 bytes, checksum: acd65707762e60b912f35a2d267ad5b7 (MD5) Previous issue date: 2016-04-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Due to the high capacity of oxygen transfer and versatility, pneumatic reactors have been constantly used in bioprocesses. However, aiming to expand the use of these bioreactors in the industry, as well as increase the understanding of the complex phenomena that occur in these devices, this thesis aimed to evaluate the hydrodynamic, oxygen transfer and shear conditions in three models of pneumatic reactors (bubble column, concentric-tube airlift and split-cylinder airlift) in the scales of 5 and 10 L, using as liquid phase four Newtonian fluids and eight non-Newtonian fluids, and five specific air flow rate (air of 1 to 5 vvm). Related to the hydrodynamic were studied the global gas hold-up (g), the gas hold-ups in the riser (R) and in the downcomer (D), liquid circulation time (tC), superficial liquid velocity in the riser (ULR) and in the downcomer (ULD), and the percentage energy losses in the riser (%ER+%EFR), in the downcomer (%ED+%EFD), and in the bottom (%EB) of airlift reactors. The values of g, R, D, ULR and ULD showed increasing behaviour with increase of air and decreasing behaviour with the kinematic liquid viscosity (L) and the rheologic properties (K e n), and observed the opposite for tC. The higher values of g, R, D and tC were obtained for concentric-tube airlift reactor (ACC) and scale of 10 L. With exception of ULR of Newtonian fluids, the others liquid velocity tests resulted in higher values for split-cylinder airlift reactors (ASC) and scale of 10 L. This result was attributed to the greater driving force (R-D) to liquid circulation obtained in the ASC reactors and the higher energy losses in the riser and in the downcomer observed in the ACC reactors. In the bottom of the airlift reactors, the higher values of %EB were obtained to the ASC reactor. To evaluate the mass transfer were studied, the average bubble diameter (Db), the volumetric oxygen transfer coefficient (kLa) and the terms that compose the kLa, the convective mass transfer coefficient (kL) and the specific interfacial area of mass transfer (aL). With the increase of air, L, K and n, the air bubbles were predominantly coalescent in water, presenting distorted shape, and non-coalescent with spherical/elliptical shape in the other solutions. It was observed a similar behavior between the kLa and aL parameters, which were directly proportional to the air and inversely proportional to the L, K and n. In water, the aL values were lower than glycerol solutions due to the higher Db values observed in this liquid. For the kL, it was observed a decreasing behaviour with the increase of the air in the most solutions. The magnitude of kL values was due mainly the oxygen difusivity in the liquid, and the higher values were observed to the water, following by the non-Newtonian solutions. In general, the higher values of the mass transfer parameters were obtained in the ACC reactor and in the scale of 10 L. The proposed method to the estimate the average shear rate velocity based on Kolmogorov’s theory of isotropic turbulence showed results consistent with the literature relative to the behavior and magnitude of this variable, as well as the results obtained by the analysis of the morphological changes of Streptomyces clavuligerus in two models of airlift reactors and two aeration conditions. Were proposed correlations to predict all evaluated parameters. Were obtained in all cases a good fit with the experimental data, with deviations between the calculated and experimental values below 20%. / Devido à alta capacidade de transferência de oxigênio e versatilidade, reatores pneumáticos têm sido constantemente utilizados em bioprocessos. Entretanto, visando ampliar a utilização destes reatores na indústria, assim como aumentar a compreensão dos fenômenos complexos que ocorrem nestes dispositivos, na presente tese teve-se como objetivo avaliar as condições hidrodinâmicas, de transferência de oxigênio e de cisalhamento em três modelos de reatores pneumáticos (coluna de bolhas, airlift de cilindros concêntricos e airlift split-cylinder) nas escalas de 5 e 10 L, utilizando como fase líquida quatro fluidos newtonianos e oito fluidos nãonewtonianos e cinco vazões específicas de ar (ar de 1 a 5vvm). Em termos hidrodinâmicos foram estudadas as retenções gasosas global (g), no riser (R) e no downcomer (D), tempo de circulação do líquido (tC), velocidade superficial do líquido no riser (ULR) e no downcomer (ULD) e as perdas percentuais de energia no riser, no downcomer e na base (%EB) de reatores airlift. Os valores de g, R, D, ULR e ULD apresentaram comportamento crescente com o aumento de ar e decrescente com a viscosidade cinemática do líquido (L) e propriedades reológicas (K e n), sendo observado o oposto para tC. Os maiores valores de g, R, D e tC foram obtidos em reator airlift de cilindros concêntricos (ACC) e escala de 10 L. Com exceção de ULR de fluidos newtonianos, os demais testes de velocidade do líquido resultaram em maiores valores nos reatores airlift split-cylinder (ASC) e escala de 10 L. Tal resultado foi atribuído a maior força motriz (R-D) para circulação do líquido obtida em ASC e às maiores perdas de energia no riser e no downcomer observadas em reatores ACC. Na base dos reatores, os maiores valores de %EB foram obtidos para reator ASC. Para avaliação da transferência de massa foram estudados o diâmetro da bolha (Db), o coeficiente volumétrico de transferência de oxigênio (kLa) e os termos que o compõe, coeficiente convectivo de transferência de massa (kL) e área interfacial específica de transferência de massa (aL). Bolhas de ar, com o aumento de ar, L, K e n foram predominantemente coalescentes em água, apresentando formato distorcido e nãocoalescentes com formato esférico/elíptico nas demais soluções. Observou-se um comportamento análogo entre kLa e aL, com relação direta à ar e inversa à L, K e n. Em água, os valores de aL foram inferiores às soluções de glicerol em virtude do maior Db observado neste líquido. Para kL, observou-se um comportamento decrescente com o aumento de ar na maioria das soluções. A magnitude dos valores de kL obedeceu principalmente a difusividade do oxigênio no líquido, sendo os maiores valores observados para água, seguido das soluções não-newtonianas. De maneira geral, os maiores valores dos parâmetros de transferência de massa foram obtidos em reator ACC de 10 L. O método proposto para estimativa da taxa de cisalhamento com base na teoria de turbulência isotrópica de Kolmogorov apresentou resultados condizentes com a literatura em termos de comportamento e magnitude desta variável, assim como com os resultados obtidos pela análise das alterações morfológicas de Streptomyces clavuligerus em dois modelos de reatores airlift e duas condições de aeração. Para todos os parâmetros avaliados foram propostas correlações para sua predição, sendo obtidos em todos os casos bons ajustes aos dados experimentais com desvios inferiores à 20%. Palavras-chave: reatores pneumáticos, retenção gasosa, kLa, diâmetro da bolha, velocidade