Thesis (Ph.D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xxiii, 248 p.; also includes graphics. Includes bibliographical references (p. 226-248). Available online via OhioLINK's ETD Center

Thesis (M.S.)--Rowan University, 2009. / Typescript. Includes bibliographical references.

Thesis (M.S.)--University of Nevada, Reno, 2005. / "August, 2005." Includes bibliographical references. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm. Online version available on the World Wide Web.

Thesis (M.S.)--University of Nevada, Reno, 2004. / "December 2004." Includes bibliographical references (leaves 59-63). Online version available on the World Wide Web.

Thesis (M.S.)--University of Nevada, Reno, 2007. / "May, 2007." Includes bibliographical references. Online version available on the World Wide Web.

Dissertation (Ph.D.)--Auburn University, 2004. / Abstract. Vita. Includes bibliographic references.

Thesis (M.S.)--Chemical Engineering, Georgia Institute of Technology, 2005. / Agrawal, Pradeep K., Committee Chair ; Bommarius, Andreas S., Committee Member ; Schork, F. Joseph, Committee Member. Includes bibliographical references.

In this study, we evaluated the potential of water-in-oil microemulsions for the separation of beta-galactosidase (lactase) from other proteins. The ability of beta-galactosidase to break down the milk carbohydrate lactose gives the enzyme considerable commercial importance. The extent of solubilization of a commercial Kluyveromyces lactis preparation of beta-galactosidase into microemulsion droplets formed from 200 mM bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was measured as a function of buffer type, pH, ionic strength, and protein concentration. Our results showed that, due to the large molecular weight of beta-galactosidase (MW~ 220-240 kDa, dimeric form), the enzyme was taken up by the microemulsion droplets mainly under very low salt conditions. Based on these results, we designed a one-step separation procedure, in which a small volume of aqueous buffer containing the protein mixture is added to an organic surfactant solution. Microemulsion droplets form in the oil and capture protein impurities of smaller molecular weights, while excluding the high molecular weight target protein. This causes the beta-galactosidase to be expelled into a newly formed aqueous phase. The feasibility of this one-step process as a bioseparation tool was demonstrated on a feed consisting of an equal mixture of beta-galactosidase and the test protein beta-lactoglobulin. Recovery and separation of the two proteins was analyzed as function of buffer type, pH, ionic strength, and protein concentration. Results showed that separation was most complete at 100 mM KCl salt concentration, where the droplets were big enough to carry beta-lactoglobulin but too small for lactase. At 100 mM salt concentration, we recovered 92% of the total lactase activity in a virtually pure form. The same separation scheme was then tested on crude extract obtained from a cell culture broth of the yeast Kluyveromyces lactis. Cells of the yeast K. lactis were disrupted by minibeadbeater, forming a crude extract that was used as the feed in our separation process. A 5.4-fold purification factor of the extract was achieved, with 96% activity recovery. The results showed our one-step separation process to be an interesting method for the production of beta-galactosidase as a technical enzyme: it has the potential to achieve a continuous, large-scale partial purification of the enzyme, potentially reducing the number of steps required in downstream process.

Orientador: Elias Basile Tambourgi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T05:30:03Z (GMT). No. of bitstreams: 1 Fischer_GilvanAnderson_D.pdf: 1330009 bytes, checksum: a03c001d3375eaae07873f0e8dd8d168 (MD5) Previous issue date: 2006 / Resumo: Bromelina é um conjunto de enzimas proteolíticas encontradas nos vegetais da família Bromeliaceae, da qual o abacaxi é o mais conhecido. A bromelina tem diversos usos, todos baseados em sua atividade proteolítica. Com a necessidade de desenvolver novos processos de purificação e concentração desses compostos a extração líquido-líquido por micela reversa mostra-se como uma técnica atrativa, pois possui a capacidade de solubilizar biomoléculas específicas de uma solução aquosa, como o extrato bruto do abacaxi. Dentre os equipamentos disponíveis para a extração líquido-líquido foi usada uma micro-coluna de campânulas pulsantes, visando a separação e purificação de biomoléculas em operação contínua. A melhor condição de extração da bromelina com micelas reversas em operação batelada foi aplicada na coluna de extração. Com o objetivo de encontrar as condições ótimas de operação da coluna foi utilizada a técnica de planejamento de experimentos do tipo estrela, de forma a abranger toda região de operação. Para isto variouse a razão Vazão Fase Leve/Vazão Total e o intervalo entre os pulsos na entrada para determinar as variações na saída do rendimento de proteína total e o fator de purificação. Os valores dos pontos ótimos para as variáveis respostas encontrados foram: fator de purificação de 5 vezes e produtividade de 1,28 mg/min. E as condições operacionais que levam ao ótimo das respostas foram: Vazão Fase Leve/Vazão Total de 0,67 e intervalo entre os pulsos de 1 s. Para representar a dinâmica de operação da coluna de extração usouse uma simples aproximação baseada nas redes neurais, pois este modelo de conexão tem a habilidade de aprender procedimentos complexos do sistema físico. As entradas foram representadas pelos passados e presentes valores da razão vazão fase leve/vazão total e no intervalo entre os pulsos, e as saídas o valor presente do rendimento de proteína total e o fator de purificação. Com o objetivo de encontrar a melhor topologia, treinou-se a rede com o algoritmo Regularização Bayesiana, variando-se o número de neurônios da camada intermediária para encontrar o melhor modelo. A topologia final da rede neural foi 16-9-2, com funções de ativação sigmoidal. Observou-se o bom desempenho da rede neural com respeito à Proteína Total e o Fator de Purificação em toda a faixa de valores desejada / Abstract: Bromelain is a set of proteolitics enzymes found in vegetables of the Bromeliaceae family, from which pineapple is known more. Bromelain has several uses, ali based on its proteolitics activity. There is the necessity to develop new processes for purification and concentration of these composites, the liquid-liquid extraction for reversed micelles reveals as one attractive technique, therefore it has had the specific capacity of getting soluble biomolecules of an aqueous solution, as the crude extract of the pineapple. Amongst equipment for the liquid-liquid extraction available it was developed a microcolumn of pulsed caps, with the purpose to get an equipment for the accomplishment of extraction processes liquid-liquid, appropriate for the separation and purification of biomolecules in continuous operation. The best condition of bromelain extraction with reversed micelles in batch operation was applied in the extraction column. To find the best operation conditions of the column, the star design of experiments was used, covering ali region of operation. For this it had been varied the ratio between low density phase to total flow and the range of time between pulses on the input to determine the variations of the total protein yield and purification factor on output each three minutes. The best values found were: 5 for purification factor and 1.28 mg/min for productivity. And operational conditions that lead to results were: 0.67 for the ratio between low-density phase to total flow and 1 s for range of time between pulses. To represent the dynamics of operation of the extraction column a simple approach based on the neural networks was used, therefore this model of connection has the ability to leam complex procedures of the physical system. The input had been represented by the pasts and presents values of the ratio between low density phase to total flow and the range of time between pulses, and the outputs the present value of the total protein yield and purification factor. To find the best topology, the network was trained with Bayesian Regularization algorithm, varying the number of neurons on hidden layer to find the best model. The final topology of the neural network was 16-9-2, with sigmoid activation functions. It was observed that the performance of the neural network is good with respect to the total protein yield and purification factor in ali range of values desired. / Doutorado / Sistemas de Processos Quimicos e Informatica / Doutor em Engenharia Química

Nanoparticles have important applications in ceramics, metal catalysts, semiconductors etc. They are normally required to be of small size (~ nm) and monodisperse. The aim of the present work is to model the formation of nanoparticles, obtained by precipitation in reverse micellar microreactors. These are dispersions of tiny water drops in a surfactant laden oil medium. Two systems were investigated: (i) Reverse micelles, having nanometer sized spherical water droplets in the micellar core and (ii) Water-in-oil emulsions, having micron-sized aqueous drops. Two modes of precipitation, namely, gas-liquid (g-1) and liquid-liquid (1-1) were studied. In each case, the models could predict the number, average size and size distribution of the particles reported in literature. Two groups have obtained widely divergent number and size of CaCO3 nanoparticles, formed by g-1 precipitation in reverse micelles. These particles are used as a fine suspension in lube-oil additives, where they serve to neutralize acid produced during combustion in engines. Kandori et al. (J. Colloid Interface Sci, 122,1988, 78) obtained particles of about 100 nm size, by passing CO2 through a reverse micellar solution, containing dissolved Ca(OH)2 in the micellar core. Roman et al. (J. Colloid Interface Sci., 144,1991, 324), instead of using lime solution; added micron-sized solid lime particles in the oil and generated the reverse micelles by in situ reaction. This is a commercial process known as overbasing. It led to a higher amount of lime in the micelles as well as unreacted lime particles in oil, at the beginning of the experiment Upon passing CO2, they got particles of only 6 nm in size, compared to 100 nm reported by Kandori et al.. Furthermore, while Kandori et al. found that one particle formed from 108 micelles, Roman et al. got one particle out of only ten micelles. We have modelled the two processes in a common framework to explain the reported disparity in particle characteristics. A time scale analysis of CO2 mass transfer, reaction, collision-fusion of micelles, nucleation, and growth of particles was carried out It showed that, in the experiments of Kandori et al., the rate limiting steps are nucleation and fusion. The analysis also indicates that the contents of a particular micelle are well mixed and reaction of lime and incoming CO2 can be treated as instantaneous. In the process of Kandori et al., the amount of lime taken initially being very small, the average number of product molecules in a micelle is well below one. Rapid Brownian coalescence and exchange of micellar contents leads to Poisson distribution of CaCO3(l) molecules formed by reaction. The low occupancy therefore suggests that most of the micelles are empty. Nucleation in a particular micelle is much slow and occurs when it has a critical number of molecules. Thus only very few micelles can nucleate. Comparison of nucleation and growth time scales - both intrinsic growth in a micelle and growth during fusion of nucleated and non-nucleated micelles - show that growth is much faster than both nucleation and collision. Hence a micelle can have only one nucleus, with subsequent growth during collisions. A population balance equation (PBE) is written involving the above steps. Solution of the moments of the distribution yields the number of CaCO3 particles, its size, coefficient of variance (COV) etc. The model not only predicts the ratio of number of micelles to particles, obtained experimentally as 108, but also captures the maxima in this quantity with increasing micellar size. The increase in average particle size with micellar size is also predicted well. The process of of Roman et ai, in addition, involves the time scale of solubilization of solid lime into micelles. Its comparison with other time scales demarcates their experiments into two distinct phases. Phase I consists of reaction of lime initially present in micelles. Time scale analysis also suggests that, as the lime content in the micelles is large, a high degree of supersaturation is rapidly generated. This results in a burst of nuclei. The other conclusions, like, well-mixed micelle, Poisson distribution of CaCO3(l) molecules, instantaneous growth and mono-nucleated micelles are found to hold good. Once the pre-existing lime is finished, relative time scales indicate that, further precipitation is controlled entirely by fresh solubilization of lime. This marks the beginning of phase II. However, solubilization being the slowest step, CaCO3(l) in micelles never builds up for any further nucleation. Phase II thus consists of pure growth of the particles formed in phase I. On developing more general PBEs and with solution of resulting moment equations - written separately for the two phases - the experimental data on number of particles and temporal evolution to the final particle size of 6 nm could be predicted very well. The model also captures the qualitative trend in COV of particle radius with time. Thus within the same framework we could successfully predict both the results, differing by seven orders of magnitude. The above analysis indicates that relative rates of nucleation, fusion-growth and mass transfer of gas controls the carbonation process. We further simplify the process and obtain an analytical solution in the limit of instantaneous mass transfer. The solution gives close first estimates for both the experiments and also indicates the smallest panicle size that could be obtained for a given experimental condition. In contrast to g-1 mode, precipitation in 1-1 mode - using two reverse micellar solutions having two reactants- occurs only on coalescence of two micelles. To obviate the solution of multivariate PBEs, we have developed a general Monte Carlo (MC) simulation scheme for nanoparticle formation, using the interval of quiescence technique (IQ). Starting with a fixed number of micelles, we conduct each coalescence-redispersion and nucleation events in this population, in the ratio of their relative frequencies. Our simulation code is much more general and realistic than the scheme of Li and Park (Langmuir, 15,1999, 952). Poisson distribution with realistic micellar occupancies of reactants, binomial redispersion of solutes after fission, a nucleation rate with critical number of molecules and Brownian collision-fusion rates were used. These considerations are based on our earlier findings in g-1 precipitation and those known in the literature too. The simulation of Li and Park then becomes a special case of our code. Our simulation code was then used to predict experimental data on two systems. The results of Lianos and Thomas (Chem. Phys. Lett. 125, 1986, 299 and /. Colloid Interface 5c/., 117, 1987, 505), on number of molecules per CdS particle, as a function of micelle size and reactant concentrations have been predicted very well. For the Fe(OH)3 nanoparticles, our simulation provides a better prediction of the experimental particle size range, than that of Li and Park. Finally, 1-1 precipitation on mixing two emulsions, having respectively the two reactants, has been simulated. Here, large reactant amount leads to multiple nucleation in a single drop and renders growth rate to be finite. This requires solving a PBE for particle population in each drop. Moreover, emulsions have a drop size distribution due to independent coalescence and breakage. The IQ technique was used for handling these events. Thus a composite model of PBE and MC for a drop population was developed. Simulation of particle size distribution in MgCO3 precipitation shows that nearly monodisperse nanoparticles can be produced in emulsions. Furthermore, average particle size can be controlled by changing reactant concentration in a drop. The findings of the thesis have provided new issues to be addressed in modelling nanoparticle formation. It points out the importance of finding models for coalescence efficiency and critical nuclear size in micelles. Extension of our model and simulation to precipitation in other organized surfactant assemblies can be done by starting from appropriate time scale analysis.