Master of Science / Department of Statistics / James J. Higgins / Completely random and randomized block designs involving n factors at each of two levels are used to screen for the effects of a large number of factors. With such designs it may not be possible either because of costs or because of time to run each treatment combination more than once. In some cases, only a fraction of all the treatments may be run. With a large number of factors and limited observations, even one outlier can adversely affect the results. Robust regression methods are designed to down-weight the adverse affects of outliers. However, to our knowledge practitioners do not routinely apply robust regression methods in the context of fractional replication of 2^n factorial treatment structures. The purpose of this report is examine how robust regression methods perform in this context.

In industrial experimentation, time and material costs are often at a premium. In designing an experiment, one needs to balance the desire for sufficient experimental runs to provide adequate data analysis, with the need to conduct a cost-effective experiment. A common compromise is the use of fractional factorial (FF) designs, in which only a fraction of the total possible runs is utilized. After discussing the basic concepts of FF designs, we introduce the fractional factorial split-plot (FFSP) design. Such designs occur frequently, because certain factors are often harder to vary than others, thus imposing randomization restric- tions. This thesis examines two techniques aimed at reducing run size that have not been greatly explored in the FFSP setting — nonregular designs and semifoldover designs. We show that these designs offer competitive alternatives to the more standard regular and full foldover designs and we produce tables of optimal designs in both scenarios.

In industrial experimentation, time and material costs are often at a premium. In designing an experiment, one needs to balance the desire for sufficient experimental runs to provide adequate data analysis, with the need to conduct a cost-effective experiment. A common compromise is the use of fractional factorial (FF) designs, in which only a fraction of the total possible runs is utilized. After discussing the basic concepts of FF designs, we introduce the fractional factorial split-plot (FFSP) design. Such designs occur frequently, because certain factors are often harder to vary than others, thus imposing randomization restric- tions. This thesis examines two techniques aimed at reducing run size that have not been greatly explored in the FFSP setting — nonregular designs and semifoldover designs. We show that these designs offer competitive alternatives to the more standard regular and full foldover designs and we produce tables of optimal designs in both scenarios.

Flow control can lead to saving millions of dollars in fuel costs each year by making an aircraft more efficient. Synthetic jets, a device for active flow control, operate by introducing small amounts of energy locally to achieve non-local changes in the flow field with large performance gains. These devices consist of a cavity with an oscillating diaphragm that divides it, into active and passive sides. The active side has a small opening where a jet is formed, whereas and the passive side does not directly participate in the fluidic jet.Research has shown that the synthetic jet behavior is dependent on the diaphragm and the cavity design hence, the focus of this work. The performance of the synthetic jet is studied under various factors related to the diaphragm and the cavity geometry. Four diaphragms, manufactured from piezoelectric composites, were selected for this study, Bimorph, Thunder®, Lipca and RFD. The overall factors considered are the driving signals, voltage, frequency, cavity height, orifice size, and passive cavity pressure. Using the average maximum jet velocity as the response variable, these factors are individually studied for each actuator and statistical analysis tools were used to select the relevant factors in the response variable. For all diaphragms, the driving signal was found to be the most important factor, with the sawtooth signal producing significantly higher velocities than the sine signal. Cavity dimensions also proved to be relevant factors when considering the designing of a synthetic jet actuator. The cavities with the smaller orifice produced lower velocities than those with larger orifices and the cavities with smaller volumes followed the same trend. Although there exist a relationship between cavity height and orifice size, the orifice size appears as the dominant factor.Driving frequency of the diaphragm was the only common factor to all diaphragms studied that was not statistically significant having a small effect on jet velocity. However along with waveform, it had a combined effect on jet velocity for all actuators. With the sawtooth signal, the velocity remained constant after a particular low frequency, thus indicating that the synthetic jet cavity could be saturated and the flow choked. No such saturation point was reached with the sine signal, for the frequencies tested. Passive cavity pressure seemed to have a positive effect on the jet velocity up to a particular pressure characteristic of the diaphragm, beyond which the pressure had an adverse effect. For Thunder® and Lipca, the passive cavity pressure that produced a peak was measured at approximately 20 and 18kPa respectively independent of the waveform utilized. For a Bimorph and RFD, this effect was not observed.Linear models for all actuators with the factors found to be statistically significant were developed. These models should lead to further design improvements of synthetic jets.

The prospect of rapid changes to the climate due to global warming is subject of public concern. The need to reduce the emissions of atmospheric green house gases and in particular carbon dioxide is greater than ever. Extensive research is performed to find new solutions and new materials, which tackles this problem in economically benign way. This thesis dealt with two potential adsorbents for post combustion  carbon capture, namely, amine modified silica and calcium carbonates. We modified porous silica with large surface area by propyl-amine groups to enhance the carbon dioxide adsorption capacity and selectivity. Experimental parameters, such as reaction time, temperature, water content, acid and heat treatment of silica substrate were optimized using a fractional factorial design. Adsorption properties and the nature of formed species upon reaction of CO2 and amine-modified silica were studied by sorption and infrared spectroscopy. Physisorbed and chemisorbed amount of adsorbed CO2 were, for the first time, estimated directly in an accurate way. The effects of temperature and moisture on the CO2 adsorption properties were also studied. Crystallization of calcium carbonate as a precursor to calcium oxide, which can be used as carbon dioxide absorbent, was studied in the second part of this thesis. Structure of different amorphous phases of calcium carbonate was studied in detail. Crystallization of calcium carbonate with and without additives was studied. Parameters like stirring rate, temperature, pH and polymer concentration showed to be important in selection of phase and morphology. An aggregation mediated crystallization was postulated to explain the observed morphologies. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Accepted.

O glicerol é gerado em grandes quantidades durante a produção de biodiesel e tem se tornado um substrato potencialmente atrativo para a produção bacteriana de produtos de valor agregado, tais como 1,3-Propanodiol (1,3-PD), etanol, hidrogênio e outros. O presente trabalho teve como objetivo a produção de hidrogênio, 1,3-PD e etanol utilizando glicerol residual obtido da síntese do biodiesel. A primeira parte do trabalho visou à produção de hidrogênio utilizando glicerol residual. Nesta etapa, o consórcio microbiano ambiental foi submetido a diferentes tratamentos (ácido, básico, choque térmico, congelamento e descongelamento e dessecação). Os resultados mostraram que a maior produção de hidrogênio ocorreu com o consórcio tratado com dessecação e choque térmico, com produções de 34,2 % mol e 27,3 % mol, respectivamente. A otimização da produção de hidrogênio foi realizada utilizando planejamento experimental e alcançou uma produção máxima de 45 % mol. A próxima etapa do projeto foi isolar as bactérias presentes no consórcio microbiano ambiental. Experimentos em biorreatores foram realizados utilizando glicerol puro e glicerol residual e os resultados mostraram que houve a completa degradação do glicerol pelas bactérias do consórcio e também a produção de 1,3-PD, hidrogênio e ácidos. Nesta etapa, 32 bactérias foram isoladas e testadas para a produção de bioprodutos em experimentos em aerobiose e anaerobiose. Quatro bactérias foram selecionadas e o seqüenciamento do gene foi identificado por 16S rRNA identificou três dos isolados como Klebsiella pneumoniae e um isolado como Pantoea agglomerans. Dentre as quatro bactérias, uma cepa, nomeada como K. pneumoniae BLh-1, foi selecionada para os trabalhos posteriores. Para otimização das condições de cultivo, utilizou-se a seleção de variáveis através do delineamento experimental Plackett Burman (P-B) avaliando a produção de 1,3-PD. Sete variáveis foram estatisticamente significativas e um novo planejamento fracionário 27-3 foi realizado com a finalidade de otimizar o meio de produção. Experimentos em biorreatores mostraram que a produção em bioreator com pH controlado com o meio otimizado no planejamento alcançou uma produção máxima de 23,80 g/L de 1,3-PD com produção de 12,30 g/L de etanol. Os ensaios com alimentação de glicerol residual mostraram que altas taxas de alimentação causam acúmulo de glicerol e diminuem a produtividade de 1,3-PD e etanol. Os estudos utilizando fluorescência 2D e modelo quimiométrico foram aplicados para avaliar as mudanças metabólicas nos cultivos aeróbios e anaeróbios. Os resultados mostraram que houve diferença nas regiões de NADH e riboflavinas ambas associadas ao metabolismo do microorganismo. / Glycerol is generated in large amounts during biodiesel production and it is becoming a potentially attractive substrate for bacterial production for value-added products such as 1,3-propanediol (1,3-PD), ethanol, hydrogen, among others. This work aimed at studying the production of hydrogen, 1,3-PD and ethanol using the raw glycerol obtained by biodiesel synthesis. The first steps were to produce hydrogen using raw glycerol. At this step, the environmental microbial consortium was submitted to different pretreatments (acid, base, heat shock, dry heat and desiccation, freezing and thawing). The results showed that dry heat and desiccation had the higher hydrogen production followed by heat shock with production of 34.2 % mol and 27.3 % mol, respectively.The optimization of hydrogen production was performed using experimental design and had a maximum hydrogen production of 45 % mol. The next spet of this project was to isolate the bacteria present in an environmental microbial consortium. Experiments in bioreactors with the microbial consortium using raw and pure glycerol were carried out and the results showed that there was a complete glycerol degradation by microbial bacteria with productions of 1,3-PD, hydrogen and acids. In this step, 32 bacteria were isolated and tested separately for by-products production under aerobic and anaerobic conditions. Four bacteria were selected and sequence gene realized by 16 S rRNA, three of them were identified as Klebsiella pneumoniae and one isolate identified as Pantoea agglomerans. Among the four bacteria, the strain K. pneumoniae BLh-1 were selected for further experiments. Plackett Burman (P-B) experimental procedure was used for assessing the production of 1,3-PD. Seven variables were statistically significant and a new 27-3 fractional design was conducted with the aim of optimizing culture conditions. Bioreactors experiments using the optimized culture conditions showed that production in bioreactor with controlled pH reached a maximal 1,3-PD production of 23.80 g/L with 12.30 g/L of ethanol. The fed-batch experiments showed that high feeding rates caused glycerol to accumulated in the medium and the1,3-PD and ethanol productivity decreased. The 2D fluorescence metodology was applied in order to study the anaerobic-aerobic changes on K. pneumoniae metabolism. The results showed a significant difference on NADH and riboflavin fluorescence that are fluorescence regions associated to metabolism.

O glicerol é gerado em grandes quantidades durante a produção de biodiesel e tem se tornado um substrato potencialmente atrativo para a produção bacteriana de produtos de valor agregado, tais como 1,3-Propanodiol (1,3-PD), etanol, hidrogênio e outros. O presente trabalho teve como objetivo a produção de hidrogênio, 1,3-PD e etanol utilizando glicerol residual obtido da síntese do biodiesel. A primeira parte do trabalho visou à produção de hidrogênio utilizando glicerol residual. Nesta etapa, o consórcio microbiano ambiental foi submetido a diferentes tratamentos (ácido, básico, choque térmico, congelamento e descongelamento e dessecação). Os resultados mostraram que a maior produção de hidrogênio ocorreu com o consórcio tratado com dessecação e choque térmico, com produções de 34,2 % mol e 27,3 % mol, respectivamente. A otimização da produção de hidrogênio foi realizada utilizando planejamento experimental e alcançou uma produção máxima de 45 % mol. A próxima etapa do projeto foi isolar as bactérias presentes no consórcio microbiano ambiental. Experimentos em biorreatores foram realizados utilizando glicerol puro e glicerol residual e os resultados mostraram que houve a completa degradação do glicerol pelas bactérias do consórcio e também a produção de 1,3-PD, hidrogênio e ácidos. Nesta etapa, 32 bactérias foram isoladas e testadas para a produção de bioprodutos em experimentos em aerobiose e anaerobiose. Quatro bactérias foram selecionadas e o seqüenciamento do gene foi identificado por 16S rRNA identificou três dos isolados como Klebsiella pneumoniae e um isolado como Pantoea agglomerans. Dentre as quatro bactérias, uma cepa, nomeada como K. pneumoniae BLh-1, foi selecionada para os trabalhos posteriores. Para otimização das condições de cultivo, utilizou-se a seleção de variáveis através do delineamento experimental Plackett Burman (P-B) avaliando a produção de 1,3-PD. Sete variáveis foram estatisticamente significativas e um novo planejamento fracionário 27-3 foi realizado com a finalidade de otimizar o meio de produção. Experimentos em biorreatores mostraram que a produção em bioreator com pH controlado com o meio otimizado no planejamento alcançou uma produção máxima de 23,80 g/L de 1,3-PD com produção de 12,30 g/L de etanol. Os ensaios com alimentação de glicerol residual mostraram que altas taxas de alimentação causam acúmulo de glicerol e diminuem a produtividade de 1,3-PD e etanol. Os estudos utilizando fluorescência 2D e modelo quimiométrico foram aplicados para avaliar as mudanças metabólicas nos cultivos aeróbios e anaeróbios. Os resultados mostraram que houve diferença nas regiões de NADH e riboflavinas ambas associadas ao metabolismo do microorganismo. / Glycerol is generated in large amounts during biodiesel production and it is becoming a potentially attractive substrate for bacterial production for value-added products such as 1,3-propanediol (1,3-PD), ethanol, hydrogen, among others. This work aimed at studying the production of hydrogen, 1,3-PD and ethanol using the raw glycerol obtained by biodiesel synthesis. The first steps were to produce hydrogen using raw glycerol. At this step, the environmental microbial consortium was submitted to different pretreatments (acid, base, heat shock, dry heat and desiccation, freezing and thawing). The results showed that dry heat and desiccation had the higher hydrogen production followed by heat shock with production of 34.2 % mol and 27.3 % mol, respectively.The optimization of hydrogen production was performed using experimental design and had a maximum hydrogen production of 45 % mol. The next spet of this project was to isolate the bacteria present in an environmental microbial consortium. Experiments in bioreactors with the microbial consortium using raw and pure glycerol were carried out and the results showed that there was a complete glycerol degradation by microbial bacteria with productions of 1,3-PD, hydrogen and acids. In this step, 32 bacteria were isolated and tested separately for by-products production under aerobic and anaerobic conditions. Four bacteria were selected and sequence gene realized by 16 S rRNA, three of them were identified as Klebsiella pneumoniae and one isolate identified as Pantoea agglomerans. Among the four bacteria, the strain K. pneumoniae BLh-1 were selected for further experiments. Plackett Burman (P-B) experimental procedure was used for assessing the production of 1,3-PD. Seven variables were statistically significant and a new 27-3 fractional design was conducted with the aim of optimizing culture conditions. Bioreactors experiments using the optimized culture conditions showed that production in bioreactor with controlled pH reached a maximal 1,3-PD production of 23.80 g/L with 12.30 g/L of ethanol. The fed-batch experiments showed that high feeding rates caused glycerol to accumulated in the medium and the1,3-PD and ethanol productivity decreased. The 2D fluorescence metodology was applied in order to study the anaerobic-aerobic changes on K. pneumoniae metabolism. The results showed a significant difference on NADH and riboflavin fluorescence that are fluorescence regions associated to metabolism.

O glicerol é gerado em grandes quantidades durante a produção de biodiesel e tem se tornado um substrato potencialmente atrativo para a produção bacteriana de produtos de valor agregado, tais como 1,3-Propanodiol (1,3-PD), etanol, hidrogênio e outros. O presente trabalho teve como objetivo a produção de hidrogênio, 1,3-PD e etanol utilizando glicerol residual obtido da síntese do biodiesel. A primeira parte do trabalho visou à produção de hidrogênio utilizando glicerol residual. Nesta etapa, o consórcio microbiano ambiental foi submetido a diferentes tratamentos (ácido, básico, choque térmico, congelamento e descongelamento e dessecação). Os resultados mostraram que a maior produção de hidrogênio ocorreu com o consórcio tratado com dessecação e choque térmico, com produções de 34,2 % mol e 27,3 % mol, respectivamente. A otimização da produção de hidrogênio foi realizada utilizando planejamento experimental e alcançou uma produção máxima de 45 % mol. A próxima etapa do projeto foi isolar as bactérias presentes no consórcio microbiano ambiental. Experimentos em biorreatores foram realizados utilizando glicerol puro e glicerol residual e os resultados mostraram que houve a completa degradação do glicerol pelas bactérias do consórcio e também a produção de 1,3-PD, hidrogênio e ácidos. Nesta etapa, 32 bactérias foram isoladas e testadas para a produção de bioprodutos em experimentos em aerobiose e anaerobiose. Quatro bactérias foram selecionadas e o seqüenciamento do gene foi identificado por 16S rRNA identificou três dos isolados como Klebsiella pneumoniae e um isolado como Pantoea agglomerans. Dentre as quatro bactérias, uma cepa, nomeada como K. pneumoniae BLh-1, foi selecionada para os trabalhos posteriores. Para otimização das condições de cultivo, utilizou-se a seleção de variáveis através do delineamento experimental Plackett Burman (P-B) avaliando a produção de 1,3-PD. Sete variáveis foram estatisticamente significativas e um novo planejamento fracionário 27-3 foi realizado com a finalidade de otimizar o meio de produção. Experimentos em biorreatores mostraram que a produção em bioreator com pH controlado com o meio otimizado no planejamento alcançou uma produção máxima de 23,80 g/L de 1,3-PD com produção de 12,30 g/L de etanol. Os ensaios com alimentação de glicerol residual mostraram que altas taxas de alimentação causam acúmulo de glicerol e diminuem a produtividade de 1,3-PD e etanol. Os estudos utilizando fluorescência 2D e modelo quimiométrico foram aplicados para avaliar as mudanças metabólicas nos cultivos aeróbios e anaeróbios. Os resultados mostraram que houve diferença nas regiões de NADH e riboflavinas ambas associadas ao metabolismo do microorganismo. / Glycerol is generated in large amounts during biodiesel production and it is becoming a potentially attractive substrate for bacterial production for value-added products such as 1,3-propanediol (1,3-PD), ethanol, hydrogen, among others. This work aimed at studying the production of hydrogen, 1,3-PD and ethanol using the raw glycerol obtained by biodiesel synthesis. The first steps were to produce hydrogen using raw glycerol. At this step, the environmental microbial consortium was submitted to different pretreatments (acid, base, heat shock, dry heat and desiccation, freezing and thawing). The results showed that dry heat and desiccation had the higher hydrogen production followed by heat shock with production of 34.2 % mol and 27.3 % mol, respectively.The optimization of hydrogen production was performed using experimental design and had a maximum hydrogen production of 45 % mol. The next spet of this project was to isolate the bacteria present in an environmental microbial consortium. Experiments in bioreactors with the microbial consortium using raw and pure glycerol were carried out and the results showed that there was a complete glycerol degradation by microbial bacteria with productions of 1,3-PD, hydrogen and acids. In this step, 32 bacteria were isolated and tested separately for by-products production under aerobic and anaerobic conditions. Four bacteria were selected and sequence gene realized by 16 S rRNA, three of them were identified as Klebsiella pneumoniae and one isolate identified as Pantoea agglomerans. Among the four bacteria, the strain K. pneumoniae BLh-1 were selected for further experiments. Plackett Burman (P-B) experimental procedure was used for assessing the production of 1,3-PD. Seven variables were statistically significant and a new 27-3 fractional design was conducted with the aim of optimizing culture conditions. Bioreactors experiments using the optimized culture conditions showed that production in bioreactor with controlled pH reached a maximal 1,3-PD production of 23.80 g/L with 12.30 g/L of ethanol. The fed-batch experiments showed that high feeding rates caused glycerol to accumulated in the medium and the1,3-PD and ethanol productivity decreased. The 2D fluorescence metodology was applied in order to study the anaerobic-aerobic changes on K. pneumoniae metabolism. The results showed a significant difference on NADH and riboflavin fluorescence that are fluorescence regions associated to metabolism.

Orientador: Francisco Maugeri Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T12:18:04Z (GMT). No. of bitstreams: 1 Aguiar-Oliveira_Elizama_D.pdf: 4155399 bytes, checksum: 664192a1e372b0f5556340a23b235a02 (MD5) Previous issue date: 2012 / Resumo: A obtenção de fruto-oligossacarídeos (FOS), um açúcar prebiótico de vasta aplicação industrial, por enzimas imobilizadas abrange dois conceitos em grande expansão: aplicação industrial de enzimas imobilizadas e alimentos funcionais. A partir de flores da Mata Atlântica Brasileira uma cepa foi isolada e identificada como Rhodotorula sp. LEB-V10 e apresentou grande potencial para produção da enzima extracelular frutosiltransferase (FTase) - capaz de produzir FOS a partir da sacarose - sendo obtida apenas por precipitação com etanol (purificação parcial) direto do meio de cultivo livre de células, de acordo com estudos realizados previamente, a cinética entre a enzima imobilizada pura e a parcialmente purificada não apresentou diferenças significativas que justificassem etapas adicionais de purificação. Estudos anteriores selecionaram a adsorção em partículas do composto sólidoácido (Nb), uma liga formada por nióbio (~95%) e grafite (~5%) como metodologia sugerida para imobilização da FTase para produção de FOS. Dessa forma, este trabalho apresenta uma série de estudos de caracterização deste biocatalisador imobilizado. Após a imobilização, foi possível observar que além do pH de máxima atividade apresentado pela enzima livre (pH 4,5) um segundo ponto distinto de máxima atividade (25% menor que em pH 4,5) também foi observado, o pH 6,0 que demonstrou também ser uma condição de maior estabilidade térmica e também melhor condição de síntese com a enzima imobilizada. O estudo da termo-estabilidade revelou também que a imobilização induziu a formação de duas zonas de energias de ativação da desnaturação (Ead) diferenciadas: entre 47 e 51°C a enzima imobilizada é mais estável que a enzima livre e entre 52 e 70°C ela é menos estável. A incubação da FTase, imobilizada e livre, em 52°C por 15 min resultou numa ativação dos biocatalisadores, sendo mais pronunciada com a enzima imobilizada e em pH 4,5 apresentando um aumento de 1,8 vezes na atividade enzimática; no entanto, observou-se que esta ativação é momentânea e não acumulativa com outras metodologias como a liofilização; isto sugere que esta temperatura representa algum tipo de limite energético para a FTase que é um hetero-dímero e pode apresentar diversas interações com o nióbio. A otimização da síntese de FOS realizada pela aplicação da metodologia de planejamento experimental com a enzima imobilizada permitiu um aumento do rendimento (YFOS = 0,58) em 5%, uma redução no tempo de síntese (24 h) em 4 vezes e um aumento da produtividade (12,05 g/L.h) em 6 vezes. A adsorção da FTase ao nióbio demonstrou não utilizar os sítios ativos uma vez que, a adição da sacarose ao meio de dispersão para adsorção, não alterou o desempenho do biocatalisador, porém a adição de CuSO4 que é um forte estabilizador térmico para esta enzima, resultou num biocatalisador incapaz de produzir FOS. Tendo em vista a aplicação industrial da FTase, avaliou-se também os efeitos da liofilização sobre a enzima livre e imobilizada. Com a enzima livre a liofilização causou perda de atividade enzimática em função da densidade da solução de partida, mas, resultou num pó com até 6 vezes mais atividade por grama do que em relação à enzima livre não liofilizada; a adição individual de compostos crio-protetores apresentou maior efeito estabilizador do que em formulações. Com a enzima imobilizada, a liofilização não apresentou grandes diferenciações, observou-se de forma geral uma manutenção das características originais do imobilizado de partida. Todavia, uma importante diferenciação proveniente da liofilização foi observada tanto para a enzima livre quanto imobilizada: a cinética de produção de FOS, o rendimento foi aumentado em diferentes proporções mas, especialmente, foi observada uma elevada composição de GF4. Estudos mais específicos sobre a estrutura tridimensional forneceriam informações mais precisas sobre o efeito da liofilização. Adicionalemente, duas enzimas comerciais (inulinase e invertase) foram igualmente imobilizadas em nióbio, mas, não foram observadas alterações tão expressivas quanto às observadas com a FTase. Dessa forma, a imobilização da FTase em nióbio revela ser uma metodologia com grande potencial de aplicação em reatores. Como resultado deste trabalho, encontram-se publicados até o momento os Capítulos 2 e 4 e aceito para publicação o Capítulo 5 / Abstract: The production of fructooligosaccharides (FOS) - a prebiotic sugar with wide industrial application - from immobilized enzymes includes two modern concepts: industrial application of immobilized enzymes and functional food. A strain isolated from flowers from the Brazilian Atlantic Forest identified as Rhodotorula sp. LEB-V10 showed great potential for production of the extracellular fructosyltransferase (FTase), which is capable of producing FOS from sucrose. It can be recovered only by precipitation with ethanol (partial purification), directly from the cell-free culture medium. According to previous studies, the kinetics between purified and partially purified immobilized enzymes showed no significant differences that could justify additional purification steps. Other previous studies have selected the adsorption on particles of a solid-acid support (Nb) an alloy consisting of niobium (~95%) and graphite (~5%) as suggested methodology for the immobilization of FTase for FOS production. Thus, this work presents a series of characterization studies of this immobilized biocatalyst. After the immobilization, it was observed that besides the pH of maximum activity presented by the free enzyme (pH 4.5), a second distinct point of maximum activity (25% lower than at pH 4.5) was also observed, pH 6.0, wich was proved to be the best condition for thermal stability and also better for synthesis with the immobilized enzyme than pH 4.5. The study of thermal stability has also shown that immobilization induces the formation of two distinct phases with different denaturation activation energy (Ead): between 47 and 51°C, the immobilized enzyme is more stable than the free enzyme, and between 52 and 70°C it is less stable. Incubation of free and immobilized FTase at 52°C for 15 min resulted in enzymatic activation, being more pronounced with the immobilized enzyme at pH 4.5 with a 1.8-fold increase in enzyme activity. However, due to other studies carried out in this work, it has been shown that this activation is merely transient and not cumulative regarding other methodologies, indicating that this temperature is a kind of energetic threshold, and also because FTase is a hetero-dimer, which may lead to different interactions with the niobium support. The optimization of the synthesis of FOS carried out by applying the methodology of experimental design with the immobilized enzyme, allowing a yield increase (YFOS = 0.58) of 5%, a reduction in synthesis time (24 h) of 4 times and an increased productivity (12.05 g/L.h) of 6 times. The adsorption on niobium has shown not to involve the FTase active sites, since the addition of sucrose to the dispersion medium for adsorption did not affect the performance of the biocatalyst. On the other hand, the addition of CuSO4, which is a strong thermal stabilizing agent for this enzyme, resulted in a biocatalyst incapable of producing FOS. Considering the industrial application of FTase, it was also evaluated the effects of lyophilization over the free and immobilized enzyme. With the free enzyme, lyophilization caused loss of enzyme activity in dependence on the density of the starting solution, but in general, resulted in a powder with up to 6 times more activity per gram as compared to non-lyophilized free enzyme; the individual effects of cryo-protectants additives presented higher stabilizing effect than with the presence of other protectants. The lyophilization of the immobilized enzyme did not show drastic effects and it was observed, in general, the maintenance of the initial properties and characteristics before lyophilization. For both, free and immobilized enzyme was possible to observe significant changes in the kinetics of FOS production, the yield was increased in different proportions but, especially, there was a high composition of GF4, which was not observed before lyophilization. In addition, two commercial enzymes (inulinase and invertase) were immobilized on niobium but no significant changes were observed as those with FTase. Thus, the immobilization of FTase in niobium proves to have a big potential for application due to operational features. As a result of this work, two articles have been published (Chapters 2 and 4) and one has been accepted for publication (Chapter 5) / Doutorado / Engenharia de Alimentos / Doutor em Engenharia de Alimentos

No description available.