A ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) faz parte da família das α-amilases e se destaca por ser a única enzima capaz de produzir ciclodextrinas (CDs). Esses oligossacarídeos cíclicos possuem a capacidade de formar complexos de inclusão com uma variedade de moléculas, alterando suas características como, por exemplo, solubilidade, volatilidade e estabilidade. Desta forma, CDs tem encontrado aplicação nas mais diversas áreas. Na indústria de alimentos, se destacam por serem potenciais estabilizantes naturais. Buscando alternativas viáveis para produção destas ciclodextrinas, neste trabalho, a enzima CGTase foi imobilizada covalentemente em esferas de quitosana e posteriormente utilizada em um reator enzimático para uso contínuo. O rendimento da imobilização foi de aproximadamente 100 %, com uma carga de 20 mg de enzima por grama de suporte seco. O processo de imobilização foi capaz de manter o comportamento da enzima frente à variação de pH e temperatura de reação, apresentando pH ótimo em 5,0 e a faixa de temperatura ótima de 70 a 95 ºC, para ambos. A estabilidade conferida ao catalisador imobilizado possibilitou sua reutilização, 61 % da sua atividade inicial foi mantida após 100 ciclos de reação. Durante utilização contínua, realizada em um reator de leito fixo, analisou-se a influência da taxa de fluxo e da concentração do substrato na geração de β-CD. A máxima produção (1,32 g / L) foi alcançada utilizando-se 4 % de amido solúvel em uma taxa de fluxo de 3 mL / min. Além disso, o biocatalisador apresentou uma ótima estabilidade operacional a 60 °C, mantendo 100 % da atividade inicial após 100 h de uso contínuo. Estes resultados demonstram que o desempenho do reator é diretamente afetado pelos parâmetros analisados e que a produção pode ser otimizada por regulação simples na velocidade de fluxo, ou pela concentração do substrato; e sugerem a possibilidade de utilizar este biocatalisador imobilizado na produção contínua de CDs. / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is member of the family α-amylase and is known for being the only enzyme able to produce cyclodextrins (CDs). These cyclic oligosaccharides have the ability to form inclusion complexes with a variety of molecules, changing its characteristics, for example, solubility, volatility and stability. Therefore, CDs have found application in several fields. In the food industry stand out for being potential natural stabilizers. Seeking to alternatives for producing these cyclodextrins, in this work, the CGTase enzyme was immobilized covalently on chitosan beads and subsequently used in enzymatic reactor for continuous use. The immobilization yield was high, reaching about 100 %, representing a load of 20 mg enzyme per gram of dry support. The immobilization process was capable of maintaining the behavior of the enzyme to the variation of pH and temperature of reaction, with pH optimum at 5.0 and the optimal temperature range of 70 - 95 ° C, for both. The stability afforded to the immobilized catalyst made possible its reuse, maintaining 61 % of its initial activity after 100 cycles of reaction. During its continuous use, in a packed bed reactor, we analyzed the influence of flow rate and concentration of the substrate in the generation of β-CD. The maximum yield (1.32 g / L) was achieved using 4 % soluble starch at a flow rate of 3 mL / min. In addition, the biocatalyst showed a great operational stability at 60 ° C, maintaining 100 % of initial activity after 100 h of continuous use. These results demonstrate that the performance is directly affected by the parameters analyzed and that the production can be optimized by simple adjustment in flow rate through the reactor, or the substrate concentration used and suggests the possibility of using this biocatalyst immobilized to the continuous production of CDs.

Packed bed combustors burn fairly large solid fuel particles within confining walls, with air supplied from below the grate. As combustion occurs and particles are consumed, fresh particles are fed onto the bed so the level is kept roughly constant. Packed bed combustion is used for wood and biomass combustion in small-scale power plants, wood waste combustion in pulp and paper plants, and trash incineration. The structure of a packed bed is very important to the combustion process and can be defined by particle shape and size, sphericity, particle overlap (decreasing area availability) and chiefly by void fraction. Void fraction has already been proven of great influence in packed beds – it is raised to the third power in the pressure loss equation, and it can also affect heat and mass transfer and surface reaction rates. This thesis presents results of several experimental combustion tests that were performed in a packed bed combustor, using commercial spruce lumber particles of parallelepipedal geometry as fuel. At the end of each test the bed contents were removed, taking care to preserve their structure, and fixed with liquefied wax. The solidified bed was then cut into circular cross sections at different heights of the bed, and photographs of the cross sections were taken so the local void fraction could be estimated using image analysis. The bed sampling led to the discovery that, surprisingly, the actual bulk void fraction in the combustor, which is the average of local void fraction measurements, is less than that of the unburnt particles, varying from 19% to 30% in decrease in void fraction depending on the particle type used. Local measurements allowed the development of an empirical linear equation model to represent the variation of void fraction with height above the grate. Each combustion test had measurements of gas volume fractions and temperatures at different heights above the bed grate to be compared with the results of a numerical model simulation. The numerical model used in this work is an existing numerical model of all the relevant processes in packed bed combustion. Previously, the numerical model had assumed the void fraction to be constant and equal to that of the unburnt fuel, since no information on local variation was available, and the packing geometry remained self-similar as particles are consumed. Three models for void fraction were then compared in the combustion model: a constant void equal to that of the unburnt particles, the empirical linear fit of void fraction with height, and a constant void equal to the measured bulk void fraction. Maximum temperatures were higher using the unburnt fuel void fraction because of a thicker oxidation zone, whereas the void fraction model iii based on experiments generated a thicker reduction zone and therefore higher CO concentrations. CO concentrations were experimentally measured and agreed quite well with the CO concentration from the model. Local void fraction differences had the most impact in the diffusion-controlled zone, as shown by comparing the empirical void model and the measured bulk void fraction. How lowering the void fraction can increase gas velocities, heat and mass transfer coefficients, and burning rates is also discussed in this work.

In this work, multiple kinetic models have been screened as potential candidates for simulating the performances of three oxidative coupling of methane (OCM) catalysts. Two of the proposed models were subjected to testing and optimization. The types of models screened covered both kinetic and microkinetic type models, i.e., radical omitting and radical considering. Some of the models only accounted for catalytic heterogeneous pathways, while others have expanded on the homogeneous gas-phase mechanism of the OCM reaction. The optimization process was carried out in MATLAB® R2020a using an error minimization tool. The range of experimental conditions examined was as follows: 740–800◦C, 100 kPa, 2–4 CH4/O2 ratio, 1–6 gcat h molC –1 spacetime. The results show successful optimization of both models as well as discrepancies in terms of their performances in predicting experimentally obtained values of CH4 and O2 conversions, as well as selectivities towards COx and C2+ products. While a kinetic model served as an easy option to optimize, it expressed limits in terms of achievable performance, mainly failing to simulate experimental runs conducted at low spacetimes. A microkinetic model on the other hand, managed to simulate all experimental conditions, with less accuracy towards COx species and much greater computational demand.

This thesis constitutes fundamental experimental and computational investigations on gasification and combustion in a packed bed of biomass. Packed bed gasification-combustion in counter-current mode is used in two applications -(1) Gasifier stove in reverse downdraft mode (or equivalently, top-lit updraft mode) that constitutes the idea behind efficient and clean burning domestic stoves. (2) Combustion-on moving grate for boiler application, studied widely in Europe. While a large part of the present study is around domestic stoves, a crucial part of the study aims to address the second application as an extension of the approach taken in the first part to clarify conflicting conclusions of earlier studies and explain the aero-thermochemical behavior over the entire range of superficial velocities, V s (this is velocity of air through the empty cross section of the reactor). Operational differences between the two applications lie in the range of superficial velocity -3.5 to 6 cm/s for domestic stoves and 15 to 30 cm/s for grate combustion. Lower values of Vs are chosen for domestic stoves to limit the particulate emissions; higher values of V s for combustion-on-grate to maximize the conversion rate. Present work deals with a fan based gasifier stove, Oorja, built by BP, India (currently transferred to FEPL, Pune) and disseminated to over 400,000 households between 2005 and 2009. The technology was developed at CGPL, IISc and transferred to BP for commercialization. Work reported in this thesis was started to resolve issues of higher CO emissions in char mode operation and occasional smoking during transition from flaming to char mode. The contribution of the thesis is split into two parts. (a) Use of the principles of gasification to improve the performance of the stoves to the highest possible level, balancing between efficiency and ash fusion issues for domestic and industrial applications and (b) fundamental studies to unravel the flame structure in the two-phase gasification-combustion process over the entire range of Vs. Improving the stove performance It has been known that in most free-convection based stoves, like three stone fire and others developed over the last two decades, the amount of energy extracted from the stove by a cooking pot, usually measured as water boiling efficiency, is between 15 to 35 % with CO emissions of more than 1.5 g/MJ. Oorja stove had demonstrated water boiling efficiency of 50 % and CO emissions of 0.75 g/MJ. Operational issues noticed in the field provided an opportunity to further improve the performance by conducting detailed thermo-chemical studies. Towards this, the components of water boiling efficiency in different phases and from different modes of heat transfer were determined. Optimizing the ratio of air flow rate between combustion air from top and gasification air through the grate (denoted by R) was the key to improving the performance. The maximum water boiling efficiency obtained was 62% with 0.53 g/MJ CO for a 320 mm diameter vessel; under these conditions, the first phase, termed flaming mode, involving pyrolysis-gasification-gas phase combustion contributed 45% to the total efficiency and 0.4 g/MJ CO at R = 4.8 and the second phase, termed char mode, involving char surface oxidation-gasification-gas phase combustion contributed 17% and 0.13 g/MJ CO at R = 1.9. Under optimal air flow conditions, efficiency depends on the size of the vessel used; reactive flow calculations were performed with fast chemistry (using mixture fraction approach) in a zone that includes the free space of the combustion chamber and the vessel to obtain the heat transfer efficiency and bring out the effect of vessel size. Experiments aimed at evaluating the performance of the stove on either side of stoichiometry, revealed that while the stove could be operated on the rich side, it was not possible to operate it on the lean side -it was always tending towards the stoichiometric point with enhanced power. Computational studies showed that increased air flow from the top caused enhanced recirculation around the fuel bed bringing more oxygen that reacted closer to the surface and transferred additional heat enhancing the pyrolysis rate, explaining the observed shift towards stoichiometry. An examination of literature showed that the energy balance for stoves had long remained unexplained (unaccounted losses in stoves were up to 40 %). Using the different components of efficiency obtained from experiments and computations, a heat balance was established to within 5%. This vast improvement in the heat balance is due to the fact that the unaccounted loss in the earlier estimates was essentially due to poor combustion, but was not so recognized. The very significant increase in combustion efficiency in this class of stoves allowed the possibility of estimating other components reasonably accurately. This is a direct consequence of the two stage gasification-combustion process yielding steady flow of gases which contain 80% (gasification efficiency) of the input energy enabling near-stoichiometric combustion with the help of controlled supply of combustion air. Fundamental studies Experiments with wood chips (615 kg/m3) and pellets (1260 kg/m3) showed that particle density has no effect on single particle and packed bed combustion in flaming mode beyond the role played through the surface energy balance (involving the product of fuel density and propagation rate, ˙r). Same is true for single char particles. A transport controlled combustion model taking into account the ash build up over the char surface confirmed this behaviour and showed that the phenomenon follows d2 law, where d is the equivalent diameter of the fuel particle, consistent with the experimental results. But packed bed of char particles showed distinct dependence on particle density. Differences were traced to poor thermal environment faced by low density wood char pieces compared to pellet char leading to variations in the volumetric heat release rate. A composite picture of the operational behaviour of the packed bed flame propagation was obtained from the measurements of exit gas composition, bed temperature, temperature of gas phase and condensed phase surface using 100 µm thermocouples, O 2 drop across the flame front using lambda sensor as a function of Vs. The packed bed studies were conducted in insulated steel and glass reactors. These studies clearly showed distinctive regimes in the bed behavior. In the first regime from Vs = 3 to 17 cm/s, (a) the propagation rate increases with Vs, (b) the fractions of CO, H2 are at least 10%, CH4 drops from 3 to 1%, (c) the oxygen fraction is near zero, (d) the gas phase temperature in the bed is constant at about 1600 K, (e) the condensed phase surface temperature increase from 850 K to 1600 K and (f) oxygen fraction drops from 0.21 to 0.0 within a single particle depth and coincides with the gas phase ignition. The inferences drawn from these data are that (i) the process is diffuusion controlled (ii) the bed operates in fuel rich mode, (iii) char participates only in reduction reactions. In the second domain from V s = 17 cm/s up to about 50 cm/s, (a) the propagation rate is nearly constant (b) the mass fractions of CO and H2 drops to less than 5%, CH4 decreases further, (c) oxygen fraction remains near zero, (d) CO 2 increases, (e) gas phase and surface temperatures are nearly equal and increase from 1600 K to 2200 K and match with the equilibrium temperature at that equivalence ratio, (f) oxygen fraction drops from 0.21 to 0 in one particle depth like in the first regime indicating diffuusion limitedness in this regime as well, (g) unlike in the first regime, volatiles from biomass are convected up to the next layer suppressing a local flame and char oxidation dominates. Beyond Vs = 50 cm/s, the propagation ceased to occur. The precise value of the extinction V s depended on the rate of increase of Vs in this range. A faster change initiated the extinction earlier. Observations showed that extinction began at some location around the periphery and spread laterally. Extinction at one layer was adequate to complete the extinction process. To explain the observed behaviour a simple zero-dimensional model tracking the heating of a fresh biomass particle upstream of the propagating flame front because of radiative heat transfer was set up. This equation was coupled with the equation for single particle flaming combustion to explain the behavior in the first regime. In order to explain the observed flattening of propagation rate in the second regime, it was found essential to account for the effect of the ash layer building on the oxidizing char particle and the temperature dependence of ash emissivity, on the radiative heat transfer to fresh biomass. The results of the model coupled with the experimental data from all sources on a corrected propagation rate vs. V s showed a universal behaviour that is considered a very important recognition of the packed bed propagation behaviour. Combining theory and experiments was essential to explain the extinction. The features are: (a) the presence of ash layer over the surface is shown to be responsible for maintaining a steady char conversion in a single particle at low stream speeds, (b) the feature that the ash layer would be blown away at stream velocities of 2.5 to 3 m/s in a single particle combustion, (c) with V s close to 50 cm/s, local velocities of air flow through the bed can reach 2 to 3 m/s, this value being sensitive to the bed arrangement (with slight shifting or settling of one particle leading to increase of the local velocity at the periphery). Thus, the high local speeds of flow through the bed (more than 2 m/s) was considered responsible for removal of ash layer such that radiation losses would be dominant and cause local extinction of the reaction front at the char surface. Thus, this study has led to a comprehensive understanding of the gasification-combustion behavior of packed bed in stoves and on grates. It has also led to the evolution of parameters for obtaining high efficiency and low emissions (HELE) from stoves -both domestic and industrial. Most interestingly, it has resulted in recognition of an universal behavior of flame propagation rate through packed bed of biomass.

O presente trabalho teve como objetivo aprimorar a síntese de biodiesel por via enzimática empregando óleo de babaçu pela rota etílica. A motivação do uso do óleo de babaçu é referente ao seu forte potencial para a produção de combustível, tendo em vista que é de baixo custo e abundante em localidades carentes de fontes de energia, o que estimula a realização de pesquisas nesta área, procurando benefícios ambientais, energéticos e econômicos. Para atingir o objetivo proposto, teve-se como proposta a transesterificação do óleo de babaçu com etanol mediada pela enzima lipase em fluxo contínuo empregando reator de leito fixo. A enzima selecionada para condução dos experimentos foi a lipase microbiana de Burkholderia cepacia (lipase PS) imobilizada covalentemente em matriz híbrida sílica-PVA com comprovada eficiência na síntese de biodiesel a partir de diferentes matérias-primas lipídicas. Todos os experimentos foram efetuados na ausência de solventes a 50?C. A parte experimental foi iniciada pelos testes comparativos de desempenho da montagem experimental proposta utilizando a preparação de lipase previamente selecionada e a lipase Novozym® 435 disponível comercialmente na forma imobilizada, de comprovada eficiência para utilização em reatores de leito fixo. Os resultados indicaram que ambas as preparações de lipase são adequadas para catalisar a síntese de biodiesel em regime contínuo, entretanto a grande limitação de operação do reator está relacionada com a elevada composição em ácidos graxos saturados (? 83%) na matéria-prima lipídica, que acarreta entupimentos nas conexões e obstrução do leito. Para contornar as limitações identificadas foi proposto um sistema de aquecimento para manter constante a temperatura na faixa requerida (45-50?C) em todo o aparato experimental. O desempenho do reator (diâmetro interno X altura: 15 mm X 210 mm) foi avaliado para substratos constituídos de óleo de babaçu e etanol em duas razões molares (1:7 e 1:12), definindo os limites de operação em termos de vazão do substrato. Foi quantificado o desempenho do sistema para 4 diferentes vazões volumétricas correspondendo a tempos espaciais entre 7 a 13h e determinadas para cada condição a influência do tempo espacial na concentração de ésteres de etila formados, rendimentos de transesterificação e produtividade. A razão molar entre os materiais de partida interferiu de maneira marcante no alcance de elevadas conversões dos ácidos graxos presentes no óleo de babaçu em seus ésteres de etila correspondentes, sendo o sistema maximizado para substratos contendo maior excesso de etanol (razão molar óleo: etanol de 1:12). Nessas condições, o funcionamento do sistema foi comprovado quantitativamente para tempos espaciais no reator iguais ou superiores a 11h, fornecendo rendimentos de transesterificação médios de 95,00 ? 4,68 % e produtividade de 54,80 ? 2,70 mgéster.gmeio-1.h-1. A lipase PS imobilizada em SiO2-PVA foi estável quanto a suas características morfológicas e catalíticas revelando tempo médio de meia-vida (t1/2) de 40 dias, sendo ainda possível sua reutilização em um novo ensaio. As características globais do desempenho do sistema contínuo proposto tornam atrativo a continuidade dos estudos em escala ampliada de processo, entretanto recomenda-se a utilização de óleos vegetais com menor teor de ácidos graxos saturados. / The purpose of this work was to enhance the enzymatic synthesis of biodiesel from babassu oil by ethanolic route. Babassu oil was chosen as a feedstock due to its potential for fuel production, including low cost and availability in regions where energy sources are needed; stimulating the developing of research projects looking for environmental, energetic and economical benefits. To attain this purpose, the transesterification reaction of babassu oil with ethanol was mediated by the enzyme lipase on packed bed reactor running on continuous mode. The selected enzyme was the microbial lipase from Burkholderia cepacia (lipase PS) covalent immobilized on hybrid matrix silica-PVA (SiO2-PVA) which showed already satisfactory performance in the biodiesel synthesis from different lipidic feedstocks. The experiments were carried out in solvent free system at 50°C. The experimental work was started by performing comparative tests of the proposed experimental assembly, using the previously selected lipase preparation and a commercial available immobilized lipase Novozym® 435, which has consistent high performance to be used on fixed packed reactors. The results indicated that both lipase preparations are able to catalyze the biodiesel synthesis under continuous operation; however limitations were found to maintain stable operation. This was related to the high concentration of saturated fatty acids (83%) present in the feedstock, which blocked the tube connections and led to bed obstruction. To overcome these limitations, a warming system was proposed to ensure constant the temperature at the required range (45-50°C) in the entirely experimental apparatus. The performance of the reactor (inner diameter X height: 15 mm X 210 mm) was evaluated for substrates composed by babassu oil and ethanol in two molar ratios (1:7 e 1:12), determining its operation limits in terms of substrate flow rate. The system performance was quantified for four different flow rates corresponded to spatial times between 7 and 13 h. For each condition, the influence of spatial times in the ethyl esters formation, transesterification yields and productivities were determined. The raw materials molar ratio interfered, in a remarkable way, the conversion of babassu oil into the correspondent ethyl esters and the system was optimized for the substrates containing high excess of ethanol (oil-to-ethanol molar ratio of 1:12). In these conditions, the reactor operation was demonstrated for spatial times higher than 11h, attaining transesterification yields of 95,00 ? 4,60% and productivities of 54,80 ? 2,70 mgesters.gmedium-1.h-1The lipase PS immobilized on SiO2-PVA was found to be stable regarding its morphological and catalytic characteristics, showing half-life time (t1/2) of 40 days. The immobilized enzyme can be also reused in a new run and the presence of substrate containing high ethanol levels did not affected the lipase stability. The performance characteristics of the proposed continuous system made attractive to develop further studies aiming at scaling up the process, however it is recommended to use vegetable oils having lower saturated fatty acids levels.

Hydrogen isotope separation systems using palladium (Pd) are currently being designed for both reactor designs with the aim of separating and purifying the reactor exhaust products which contain valuable unspent hydrogen isotopes. Hydrogen isotope exchange in Pd offers an efficient, ambient condition process that can produce pure isotopic species in a process far simpler and less costly than the current state of the art cryogenic distillation processes. The method is applicable whether separating hydrogen (protium), deuterium or tritium and any combination of these. If practical fusion devices are ever to be realised it is essential to produce an economical and efficient fuel cycle capable of separating and purifying hydrogen isotopes. Hydrogen isotope exchange in Pd is also of interest to the waste separation and purification industries, in particular those using hydrogen separation membranes which used Pd and Pd-alloy membranes. Understanding hydrogen isotope exchange, with particular regard to the formation of the intermediate (and often unwanted) hydrogen deuteride (HD), will aid significantly in future designs of hydrogen isotope separation systems. Novel hydrogen isotope exchange experiments involving hydrogen and deuterium at a number of temperatures (208 K, 293 K and 373 K) and pressures (1.3 bar – 8 bar) not yet explored are presented in this thesis. The experiments were carried out on a unique piece of laboratory apparatus provided to and further developed at the University of Bath. Alongside experimentation, a novel comprehensive multidimensional multi-physics model has been created to analyse the experimental data obtained using the new apparatus and elucidate the kinetics and mechanisms of the reactions occurring between hydrogen isotopic species and Pd during hydrogen isotope exchange based on Langmuir-Hinshelwood surface reaction mechanism. The surface reaction rates, kinetic rate constants and heat effects have been examined in detail, and in tandem, for the first time.

Computational Fluid Dynamics (CFD) is one of the fields that has strongly developed since the recent development of faster computers and numerical modeling. CFD is also finding its way into chemical engineering on several levels. We have used CFD for detailed modeling of heat and mass transfer in a packed bed. One of the major questions in CFD modeling is whether the computer model describes reality well enough to consider it a reasonable alternative to data collection. For this assumption a validation of CFD data against experimental data is desired. We have developed a low tube to particle, structured model for this purpose. Data was gathered both with an experimental setup and with an identical CFD model. These data sets were then compared to validate the CFD results. Several aspects in creating the model and acquiring the data were emphasized. The final result in the simulation is dependent on mesh density (model detail) and iteration parameters. The iteration parameters were kept constant so they would not influence the method of solution. The model detail was investigated and optimized, too much detail delays the simulation unnecessarily and too little detail will distort the solution. The amount of data produced by the CFD simulations is enormous and needs to be reduced for interpretation. The method of data reduction was largely influenced by the experimental method. Data from the CFD simulations was compared to experimental data through radial temperature profiles in the gas phase collected directly above the packed bed. It was found that the CFD data and the experimental data show quantitatively as well as qualitatively comparable temperature profiles, with the used model detail. With several systematic variances explained CFD has shown to be an ample modeling tool for heat and mass transfer in low tube to particle (N) packed beds.

Visando ampliar as rotas de produção de biodiesel, o objetivo deste trabalho foi utilizar uma tecnologia alternativa para a produção de biodiesel por via enzimática. Neste processo, células do fungo Mucor circinelloides URM 4182 foram cultivadas em meio apropriado, imobilizadas in situ em espuma de poliuretano e utilizadas diretamente como biocatalisador na síntese de ésteres de ácidos graxos em reações de transesterificação do óleo de coco usando etanol como agente acilante. O trabalho experimental foi desenvolvido em 4 etapas. Inicialmente, as propriedades bioquímicas do biocatalisador foram determinadas, incluindo valores ótimos de pH (7,3) e temperatura (40° C) e constantes cinéticas (Vmax= 610 U/g e Km= 263 mM). Na segunda etapa foram realizados testes utilizando reatores de tanque agitados operados em regime descontínuo confirmando a eficiência do M. circinelloides UMR 4182 para converter os ácidos graxos presentes no óleo de coco em ésteres correspondentes (rendimento de transesterificação da ordem de 96% em 144 horas). Com objetivo de aumentar a produtividade do sistema, na terceira etapa, estudos foram conduzidos em reator de leito fixo operando em fluxo contínuo empregando dois reatores (A e B) com diferentes relações geométricas altura/diâmetro (l/d=4,2 e l/d=14). Avaliou-se inicialmente no reator A o comportamento do sistema para substratos preparados em diferentes razões molares (1:6, 1:8 e 1:10 óleo/álcool). Comparativamente os rendimentos mais elevados (da ordem de 62%) foram obtidos para substratos preparados na razão molar de 1:8 (óleo/álcool), indicando a necessidade de investigar outros parâmetros do processo. A presença de caminhos preferenciais detectada visualmente no reator A pela injeção do corante lipossolúvel indicou limitações de transferência de massa, justificando a baixa conversão em ésteres de etila. Em seguida, mediante a avaliação da geometria dos reatores, foi selecionado o reator B (volume útil - 286 mL) para determinar a influencia do tempo espacial (60, 70 e 80 horas) no desempenho do processo utilizando a condição pré-estabelecida, etanol em excesso na razão molar 1:8 (óleo/etanol). O melhor desempenho do reator foi constatado para tempo espacial de 80h (vazão volumétrica = 0,06 mL.min-1). Nessa condição, 92,7 ??1,5% dos ácidos graxos presentes no óleo de coco foram convertidos nos ésteres de etila correspondentes, alcançando uma produtividade média de 51,0 ??1,4 mgéster.g-1meio.h-1 e fornecendo uma operação estável do sistema por 25 dias. As amostras purificadas apresentaram valores de viscosidade 5,14 mm2/s e teores residuais de monoglicerídeos (3,2 m/m%) e diglicerídeos (3,4 m/m%). Na quarta etapa foram realizados testes em reatores agitados operados em regime descontínuo na presença de solvente (terc-butanol e/ou líquido iônico), em diferentes proporções (10 - 50%). Os resultados foram comparados com a reação conduzida na ausência de solvente que apresentou baixa velocidade de reação e rendimento reduzido (44,5% em 6 dias de reação). Ambos os solventes aumentaram as conversões e velocidades de reação; no entanto, terc-butanol apresentou melhor desempenho em menor proporção (15%). Nessa condição, rendimentos da ordem de 96% foram alcançados em 96 horas, provavelmente devido à polaridade moderada e baixa viscosidade deste solvente que dilui o glicerol acumulado no suporte e melhora a transferência do substrato para o interior das células imobilizadas, impedindo a inibição da enzima por limitação da transferência de massa. De forma geral, os resultados foram promissores e demonstraram a potencialidade das células íntegras de M. circinelloides URM 4182 imobilizada em poliuretano para mediar a síntese de biodiesel em um reator de leito fixo em fluxo contínuo. / Aiming at expanding routes for biodiesel production, the goal of this work was to use an alternative technology for the production of biodiesel by enzymatic route. In this case, whole-cells from filamentous fungus Mucor circinelloides URM 4182 were growth in appropriate medium, simultaneously immobilized on low-cost support and used directly as a biocatalyst in the transesterification reactions of coconut oil using ethanol as the acylating agent. The experimental work was carried out in four steps. Initially, the biochemical properties of the immobilized biocatalyst were determined, including values for optimum pH (7.3) and temperature (40 °C) and kinetics parameters (Vmax 610 U/g and Km= 263 mM). Following this, tests were carried out in stirred batch reactors confirming the efficiency of M. circinelloides URM 4182 to produce biodiesel from coconut oil (96% in 144 hours). However, the low productivity attained motivated the replacement of the batch system for packed bed reactor operating under continuous flow using two reactor (A and B) having different geometric relations height/diameter (l/d = 4.2 and l/d = 14). Initially, the performance of the reactor A was assessed for substrates at different molar ratios (1:6, 1:8 and 1:10 oil/alcohol). Comparatively the best conversion (62%) was achieved for substrate at molar ratio of 1:8 (oil/alcohol); suggesting the need to investigate other process parameters to overcome such limitations. The presence of preferential paths visual detected for Reactor A by tracer assay indicated severe mass transfer limitations, justifying the low conversion attained in ethyl esters. Following this, Reactor B was selected to run the continuous experiments using substrate at pre-established condition (molar ratio oil/ethanol 1: 8) under different space times (60, 70, and 80 hours). Better reactor performance was found for space time of 80 h (volumetric flow = 0.06 mL min-1). In this condition, 92.7±1.5% of the fatty acids present in the coconut oil have been converted into the corresponding ethyl esters, reaching an average volumetric productivity of 51.0 ??1.4 mgester.g-1medium.h-1 with no significant reduction in the efficiency during 25 days. The purified samples presented values of 5.14 viscosity mm2/s and residual levels of monoglycerides (3.2 wt %) and diglycerides (3.4 wt%). In the fourth step, transesterification reactions were performed in stirred tank reactors in the presence of solvent (tert-butanol and/or Ionic Liquid) at different proportions (10-50%) in relation to the total weight oil and ethanol. The results were compared with the reaction carried out in the absence of solvent which has low reaction rate and reduced yield (44.5% in 6 days of reaction). Both solvents enhanced the transesterification yields and reaction rates; however tert-butanol showed better performance at lower proportion (15%). Under this condition, yields in the order of 96% were attained in 96 hours, probably due to the moderate polarity and lower viscosity of this solvent which dilutes the glycerol accumulated in support and improved the substrate transfer into the interior of the immobilized cells by preventing the enzyme inhibition by limitation of the mass transfer. Overall, the results were promising and showed the potential of whole-cells from M. circinelloides URM 4182 immobilized in polyurethane to mediate the synthesis of biodiesel in a continuous packed bed reactor.

O presente trabalho teve como objetivo aprimorar a síntese de biodiesel por via enzimática empregando óleo de babaçu pela rota etílica. A motivação do uso do óleo de babaçu é referente ao seu forte potencial para a produção de combustível, tendo em vista que é de baixo custo e abundante em localidades carentes de fontes de energia, o que estimula a realização de pesquisas nesta área, procurando benefícios ambientais, energéticos e econômicos. Para atingir o objetivo proposto, teve-se como proposta a transesterificação do óleo de babaçu com etanol mediada pela enzima lipase em fluxo contínuo empregando reator de leito fixo. A enzima selecionada para condução dos experimentos foi a lipase microbiana de Burkholderia cepacia (lipase PS) imobilizada covalentemente em matriz híbrida sílica-PVA com comprovada eficiência na síntese de biodiesel a partir de diferentes matérias-primas lipídicas. Todos os experimentos foram efetuados na ausência de solventes a 50?C. A parte experimental foi iniciada pelos testes comparativos de desempenho da montagem experimental proposta utilizando a preparação de lipase previamente selecionada e a lipase Novozym® 435 disponível comercialmente na forma imobilizada, de comprovada eficiência para utilização em reatores de leito fixo. Os resultados indicaram que ambas as preparações de lipase são adequadas para catalisar a síntese de biodiesel em regime contínuo, entretanto a grande limitação de operação do reator está relacionada com a elevada composição em ácidos graxos saturados (? 83%) na matéria-prima lipídica, que acarreta entupimentos nas conexões e obstrução do leito. Para contornar as limitações identificadas foi proposto um sistema de aquecimento para manter constante a temperatura na faixa requerida (45-50?C) em todo o aparato experimental. O desempenho do reator (diâmetro interno X altura: 15 mm X 210 mm) foi avaliado para substratos constituídos de óleo de babaçu e etanol em duas razões molares (1:7 e 1:12), definindo os limites de operação em termos de vazão do substrato. Foi quantificado o desempenho do sistema para 4 diferentes vazões volumétricas correspondendo a tempos espaciais entre 7 a 13h e determinadas para cada condição a influência do tempo espacial na concentração de ésteres de etila formados, rendimentos de transesterificação e produtividade. A razão molar entre os materiais de partida interferiu de maneira marcante no alcance de elevadas conversões dos ácidos graxos presentes no óleo de babaçu em seus ésteres de etila correspondentes, sendo o sistema maximizado para substratos contendo maior excesso de etanol (razão molar óleo: etanol de 1:12). Nessas condições, o funcionamento do sistema foi comprovado quantitativamente para tempos espaciais no reator iguais ou superiores a 11h, fornecendo rendimentos de transesterificação médios de 95,00 ? 4,68 % e produtividade de 54,80 ? 2,70 mgéster.gmeio-1.h-1. A lipase PS imobilizada em SiO2-PVA foi estável quanto a suas características morfológicas e catalíticas revelando tempo médio de meia-vida (t1/2) de 40 dias, sendo ainda possível sua reutilização em um novo ensaio. As características globais do desempenho do sistema contínuo proposto tornam atrativo a continuidade dos estudos em escala ampliada de processo, entretanto recomenda-se a utilização de óleos vegetais com menor teor de ácidos graxos saturados. / The purpose of this work was to enhance the enzymatic synthesis of biodiesel from babassu oil by ethanolic route. Babassu oil was chosen as a feedstock due to its potential for fuel production, including low cost and availability in regions where energy sources are needed; stimulating the developing of research projects looking for environmental, energetic and economical benefits. To attain this purpose, the transesterification reaction of babassu oil with ethanol was mediated by the enzyme lipase on packed bed reactor running on continuous mode. The selected enzyme was the microbial lipase from Burkholderia cepacia (lipase PS) covalent immobilized on hybrid matrix silica-PVA (SiO2-PVA) which showed already satisfactory performance in the biodiesel synthesis from different lipidic feedstocks. The experiments were carried out in solvent free system at 50°C. The experimental work was started by performing comparative tests of the proposed experimental assembly, using the previously selected lipase preparation and a commercial available immobilized lipase Novozym® 435, which has consistent high performance to be used on fixed packed reactors. The results indicated that both lipase preparations are able to catalyze the biodiesel synthesis under continuous operation; however limitations were found to maintain stable operation. This was related to the high concentration of saturated fatty acids (83%) present in the feedstock, which blocked the tube connections and led to bed obstruction. To overcome these limitations, a warming system was proposed to ensure constant the temperature at the required range (45-50°C) in the entirely experimental apparatus. The performance of the reactor (inner diameter X height: 15 mm X 210 mm) was evaluated for substrates composed by babassu oil and ethanol in two molar ratios (1:7 e 1:12), determining its operation limits in terms of substrate flow rate. The system performance was quantified for four different flow rates corresponded to spatial times between 7 and 13 h. For each condition, the influence of spatial times in the ethyl esters formation, transesterification yields and productivities were determined. The raw materials molar ratio interfered, in a remarkable way, the conversion of babassu oil into the correspondent ethyl esters and the system was optimized for the substrates containing high excess of ethanol (oil-to-ethanol molar ratio of 1:12). In these conditions, the reactor operation was demonstrated for spatial times higher than 11h, attaining transesterification yields of 95,00 ? 4,60% and productivities of 54,80 ? 2,70 mgesters.gmedium-1.h-1The lipase PS immobilized on SiO2-PVA was found to be stable regarding its morphological and catalytic characteristics, showing half-life time (t1/2) of 40 days. The immobilized enzyme can be also reused in a new run and the presence of substrate containing high ethanol levels did not affected the lipase stability. The performance characteristics of the proposed continuous system made attractive to develop further studies aiming at scaling up the process, however it is recommended to use vegetable oils having lower saturated fatty acids levels.

A ciclodextrina glicosiltransferase (CGTase) é a única enzima capaz de catalisar a reação de ciclização a partir do amido e, assim, formar oligossacarídeos cíclicos conhecidos como ciclodextrinas (CDs). Através desta reação é produzida uma mistura de α-, β- e γ-CD que, respectivamente, contém 6, 7 e 8 resíduos de glicose. As CDs têm atraído enorme atenção devido ao seu grande potencial de aplicação em diversas áreas da indústria. Potencial este proporcionado por sua estrutura cônica, com interior hidrofóbico, capaz de encapsular sólidos, líquidos e gases, conferindo propriedades importantes e protegendo-os. Neste trabalho foi estudada a imobilização de uma CGTase em sílica mesoporosa de forma direcionada às cisteínas presentes em sua superfície, alterando a exposição do sítio ativo. A ligação via cisteínas nativas da proteína aumentou em quatro vezes a eficiência da imobilização, quando comparada a ligação via grupamento amino. Esta, no entanto, apresentou maior atividade enzimática em faixas mais amplas de temperatura e pH, além de maior estabilidade operacional, mantendo 100 % de sua atividade após 200 h de reação contínua a 60 °C e pH 4. Ainda que apresentando menor estabilidade da ligação, o derivado obtido por ligação dissulfeto manteve 40 % da atividade inicial durante 200 h e então, o suporte pôde ser recarregado e reutilizado por igual período. Os suportes desenvolvidos apresentaram estabilidade satisfatória, possibilitando o uso do derivado imobilizado em reator de leito fixo operado de forma contínua. Quando avaliado em relação a produção das três ciclodextrinas principais, o derivado cuja imobilização da enzima ocorreu via grupamento amino, evidenciou a possibilidade de modulação da produção apenas variando as condições de reação. α- e β-CD foram produzidas preferencialmente em pH 8,0 e 2 min (3,44 mg mL-1 e 3,51 mg mL-1, respectivamente), enquanto que pH mais ácido (4,0) e maior tempo de reação (141 min) favoreceram a formação de γ-CD (3,35 mg mL-1), com baixa formação α-CD (0,75 mg mL-1). Por fim, os resultados deste estudo evidenciam a importância da imobilização da CGTase para a estabilização de sua estrutura a fim de aplicá-la em sistemas contínuos de produção de CDs onde é possível modular o perfil dos produtos gerados em função das condições de reação, aumentando assim a produtividade do biocatalisador. / Cyclodextrin glycosyltransferase (CGTase) is the only enzyme capable of catalyzing the cyclization reaction from the starch and thus forming cyclic oligosaccharides known as cyclodextrins (CDs). Through this reaction, is produced a mixture of α-, β- and γ-CD containing, 6, 7 and 8 glucose residues respectively. Cyclodextrins (CD) have been attracting considerable attention because of its great potential for application in various areas of industry. This potential is provided by its conical structure with hydrophobic interior, capable of encapsulating solids, liquids and gases, changing important features and protecting them. In this work, the immobilization of CGTase in mesoporous silica was studied in a way directed to cysteines present on its surface, altering the exposure of the active site. The connection via native cysteine of the protein increased by four times the efficiency of immobilization compared to amino groups connection. The binding of amino groups, however, showed greater enzymatic activity in wider ranges of temperature and pH, and higher operational stability, while maintaining 100 % of its activity after 200 h of continuous reaction at 60 °C and pH 4. Although showing less stable connection, the derivative obtained by disulfide bond retained 40 % of the initial activity for 200 h and then, the support could be reloaded and reused for the same period. Developed supports showed satisfactory stability, enabling the use of the derivative assets in a packed bed reactor and operated continuously. It was demonstrated the possibility of modulating the CDs production just varying the reaction conditions, using the derivative of which the enzyme immobilization occurred via amino group, to evaluate the production of three main cyclodextrins. α- and β-CD were produced preferentially at pH 8.0 and 2 min (3.44 mg mL-1 and 3.51 mg mL-1, respectively), whereas the more acid pH (4.0) and longer reaction (141 min) favored the formation of γ-CD (3.35 mg mL-1 and 0.75 mg mL-1 of α-CD). Finally, the results of this study show the importance of the immobilization of CGTase to the stabilization of its structure in order to apply it in continuous CD production systems, where it is possible to modulate the profile of the products generated as a function of the reaction conditions, thus increasing the productivity of the biocatalyst.