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.

Biodiesel is the most successful drop-in biofuel used in transportation. It can reduce GHG emissions in transportation by 50 to 90% depending on the type of feedstock used. Waste cooking oils and fats containing free fatty acids (FFA) are less expensive feedstocks for biodiesel production than refined vegetable oils. The major issue that limits the use of these oils as feedstock is the interference of FFAs with widely used base catalyzed reaction processes. The FFAs consume base catalyst, produce water of neutralization and form soaps that create emulsions downstream in the process reducing process yields. There is an important need to develop technologies that reduce the FFA content in these oils to below 0.5 wt%; the accepted limit for a feedstock to be processed by the base catalysed reaction. Enzymes are an efficient and environmentally friendly catalyst for FFA esterification. However, they are prone to deactivation with methanol and also catalyze the hydrolysis of esters and triglycerides to FFA. Using them to pre-treat oils and fats remains a challenge: in the presence of water, enzymes can readily produce FFAs from lipids. The objective of this work was to investigate two enzymatic processes to pre-treat acidic oil below the FFA requirement of 0.5 wt%. In this study, two different continuous systems, a packed bed reactor (PBR) and membrane reactor (MR) were used in FFA enzymatic esterification to meet the 0.5 wt% requirement, improve the reusability of enzymes and reduce catalyst cost. The esterification in the PBR was carried out using CALB immobilized on a new 2D/3D nanoplatelet support (TAN). The enzyme was covalently bonded to the TAN using a hydrophobic epoxy ligand. Acidic oil containing canola oil and 2.5 wt% FFA was used as the feedstock for the esterification. It was found that the FFA concentration met the quality specification of <0.5 wt% using CALB-TAN, while it did not using the commercial Novozym 435. The surface fluid velocity was found to have an effect on the removal of water from the PBR reactor. When the velocity was too low, water was retained in the reactor and the FFA conversion was low, when it was too high the reaction time for esterification was not sufficient. It was found that feed velocity of 3 to 6 x 10-5 m/s met the 0.5 wt% requirement. In the PBR, the use of CALB-TAN successfully eliminated the hydrolysis of TG and achieved the continuous esterification of FFA for 42 days. In the MR, acidic oil containing canola oil and 10 wt% FFA was used as the feedstock for the esterification. The enzyme adsorbed on the surface of the polar phase containing glycerol and water and was successfully retained in the reactor by a 0.2-micron ceramic membrane. The addition of glycerol increased the polarity of the dispersed phase in the reactor, bounded water, and retained the liquid enzyme in the reactor. However, the added glycerol in the reactor increased the operating pressure of the reactor. The operating pressure was reduced by adding biodiesel to the feedstock prior to treatment. The lowest level of FFA from the 10 wt% FFA feedstock was 0.68 wt%. This would require a second polishing step to reach the required 0.5 wt%. The PBR and MR using CALB are technologies that limit the hydrolysis at low FFA concentrations and are promising for the pre-treatment of acidic feedstocks in base catalysed biodiesel processes.

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.

abstract: Arsenic (As) is a naturally occurring element that poses a health risk when continually consumed at levels exceeding the Environmental Protection Agencies (EPA) maximum contaminant level (MCL) of 10 ppb. With the Arizona Department of Water Resources considering reliance on other sources of water other than just solely surface water, groundwater proves a reliable, supplemental source. The Salt River Project (SRP) wants to effectively treat their noncompliance groundwater sources to meet EPA compliance. Rapid small-scale column tests (RSSCTs) of two SRP controlled groundwater wells along the Eastern Canal and Consolidated Canal were designed to assist SRP in selection and future design of full-scale packed bed adsorbent media. Main concerns for column choice is effectiveness, design space at groundwater wells, and simplicity. Two adsorbent media types were tested for effective treatment of As to below the MCL: a synthetic iron oxide, Bayoxide E33, and a strong base anion exchange resin, SBG-1. Both media have high affinity toward As and prove effective at treating As from these groundwater sources. Bayoxide E33 RSSCT performance indicated that As treatment lasted to near 60,000 bed volumes (BV) in both water sources and still showed As adsorption extending past this operation ranging from several months to a year. SBG-1 RSSCT performance indicated As, treatment lasted to 500 BV, with the added benefit of being regenerated. At 5%, 13%, and 25% brine regeneration concentrations, regeneration showed that 5% brine is effective, yet would complicate overall design and footprint. Bayoxide E33 was selected as the best adsorbent media for SRP use in full-scale columns at groundwater wells due to its simplistic design and high efficiency. / Dissertation/Thesis / Masters Thesis Civil and Environmental Engineering 2015

Made available in DSpace on 2014-06-11T19:24:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:21:12Z : No. of bitstreams: 1 casciatori_fp_me_sjrp_parcial.pdf: 81692 bytes, checksum: 8909085df78fb65d202e35e91ecbb5b2 (MD5) Bitstreams deleted on 2015-08-28T16:08:54Z: casciatori_fp_me_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-08-28T16:09:59Z : No. of bitstreams: 1 000640118.pdf: 1821375 bytes, checksum: 2ac04413db80f0e987e207401ed80680 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O propósito deste trabalho foi obter parâmetros físicos e térmicos para simulação de reatores de leito fixo para fermentação em estado sólido (FES), empregando rejeitos sólidos agroindustriais como substratos e fungos como agentes fermentativos. Os materiais estudados foram bagaço de cana, resíduo agroindustrial comumente empregado na FES, e um meio composto por bagaços de cana e laranja e farelo de trigo na proporção 1:2:2 (p/p), substrato empregado para obtenção de enzimas pectinolíticas por FES. Foi determinada a condutividade térmica na estagnação (K0) pelo método da sonda linear. Observou-se que a condutividade era muito baixa para os recheios secos (inferior a 0,1 W/mºC), mas aumentava bruscamente para recheios muito úmidos (atingindo 0,5 W/mºC), o que foi atribuído à condução de calor através da água. Também foram determinados os parâmetros térmicos dos leitos percolados por ar com baixas vazões, típicas da FES. Os valores da condutividade térmica efetiva radial (Kr) obtidos foram muito baixos, tendendo ao valor da estagnação, e os valores do coeficiente convectivo de transferência de calor parede-fluido (hp) foram muito inferiores aos encontrados na literatura, devido às baixas vazões de ar. Foram desenvolvidos programas para aquisição de dados experimentais de temperatura por meio do software de interface gráfica LabView (National Instruments). Também foram desenvolvidos programas em MatLab para cálculo dos parâmetros térmicos dos sistemas, tanto para o modelo unidimensional (coeficiente global U) quanto para o bidimensional. Esses programas permitiram calcular os parâmetros térmicos imediatamente após aquisição dos dados do processo. Programas de simulação numérica foram desenvolvidos em MatLab para simular o crescimento do fungo termofílico Thermomucor indicae seudaticae N31 e o comportamento térmico do processo de FES... / The purpose of this work was to get physical and thermal parameters for simulation of reactors of fixed packed beds percolated by air for solid state fermentation (SSF), using agro- industrial solid residues as substrata and molds as fermentative agents. The materials studied were sugar cane bagasse, agro-industrial residue commonly used in SSF, and a medium composed by sugar cane and orange bagasses and wheat bran in ratio 1:2:2 (w/w), substratum used for pectinolitics enzymes attainment for SSF. The thermal conductivity on stagnation (K0) was determined by heating linear method. It was observed that the conductivity was very low for dried materials (below 0,1 W/mºC), but increased brusquely for very humid materials (reaching 0,5 W/mºC), what it was admitted to be due to conduction of heat through the water. Also the thermal parameters of the fixed packed beds percolated by air with low outflows, typical of SSF, had been determined. The values of the thermal conductivity radial effective (Kr) gotten had been very low, tending to the value of the stagnation, and the values of the convective coefficient of heat transfer wall-fluid (hp) had been very below to the found on literature, which had to the low air outflows. Programs for acquisition of experimental data of temperature by means of the software of graphical interface LabView had been developed (National Instruments). Also programs in MatLab for calculation of the thermal parameters of the systems had been developed, as much for the one-dimensional model (global coefficient U) how much for the two-dimensional. These programs had allowed calculating the thermal parameters immediately after acquisition of the data of the process. Programs of numerical simulation had been developed in MatLab to simulate the growth of thermopile mold Thermomucor indicae seudaticae N31 and the thermal behavior of the process of SSF lead by Umsza Guez... (Complete abstract click electronic access below)

Trickle flow is encountered in a variety of process engineering applications where gas and liquid flow through a packed bed of stationary solid. Owing to the complexities of three interacting phases, a fundamentally exhaustive description of trickle flow hydrodynamics has not been achieved. A complicating factor in describing the hydrodynamics is the fact that the hydrodynamic state is dependent not only on the present operating conditions but also on their entire history, including fluid flow rate changes and pre-wetting procedures. This phenomenon is termed hydrodynamic multiplicity and is the subject of this work. Hydrodynamic multiplicity greatly complicates both the experimental investigation into the behaviour of a trickle flow column and the theoretical modelling of the observed behaviour. Broadly speaking, this study addresses hydrodynamic multiplicity on three levels. First, a conceptual framework is proposed that can be used to study hydrodynamic multiplicity with limited resources. It is based on the absolute limiting values that the hydrodynamic parameters can adopt for a certain set of conditions, and encompasses both flow rate hysteresis loops and pre-wetting procedures. There are 5 such hydrodynamic modes. When the existing literature is critically evaluated in light of this framework, it is established that the reported experimental studies have not addressed all the issues. Previous modelling attempts are also shown to be unable to qualitative explain all the existing data. Moreover, authors have suggested different (and often contradictory) physical mechanisms responsible for hydrodynamic multiplicity. Secondly, an experimental investigation intended to supplement the existing literature and illustrate the utility of the proposed framework is launched. This includes bed-scale measurements of liquid holdup, pressure drop and gas-liquid mass transfer for a variety of conditions including different flow rates, pressures, particle shapes, particle porosity and surface tension. The second part of the experimental effort uses radiography and tomography in new ways to visualise the temporal and spatial characteristics of the different hydrodynamic modes. The tomographic investigation incorporates advanced image processing techniques in order to culminate in a pore-level evaluation of the hydrodynamic modes that reveals additional features of hydrodynamic multiplicity. Thirdly, the experimental insights are condensed into a set of characteristic trends that highlight the features of hydrodynamic multiplicity. A pore-level capillary mechanism is then introduced to qualitatively explain the observed behaviour. The mechanism shows how the differences in advancing and receding contact angles and the characteristics of the packed structure (or pore geometries) are ultimately responsible for the observed hydrodynamic multiplicity behaviour. Lastly, the effect of hydrodynamic multiplicity on trickle bed reactor performance is discussed. It is established experimentally that depending on the reaction conditions, different modes yield optimal performance. The idea of optimizing the performance by manipulating the hydrodynamic state is introduced. In totality, this work advances the understanding of trickle flow hydrodynamics in general and hydrodynamic multiplicity in particular. / Thesis (PhD (Chemical Engineering))--University of Pretoria, 2008. / Chemical Engineering / unrestricted

Wastewater treatment performance of three pilot-scale packed-bed anaerobic digesters with walnut shell medium was researched for treating dairy freestall barn flushwater. Reciprocation mixing was evaluated as a means to lessen channelization in the media bed and to improve biogas production and organic matter removal at ambient temperatures. Reciprocation has been used in biological nitrogen removal systems to introduce air into the system to repeatedly oxygenate nitrifying biofilm along with mixing (Behrends et al. 2003), but the anaerobic systems benefit from mixing. Two tanks were used in each system, where one was full and one was empty at any given time. Water was repeatedly pumped from one tank to the other and back again (reciprocation). A key research objective was to determine the minimum reciprocation frequency (between 0-10 per day) while still maintaining moderate methane production and treatment performance. Broken walnut shells with a specific surface area of 360 m2/m3 were used as the packed media. Digester influent, which was pretreated to remove large solids, had the following characteristics: total solids (TS) of 5.5 g/L, volatile solids (VS) of 2.8 g/L, 5-day carbonaceous biochemical oxygen demand (cBOD5) of 800 mg/L, and chemical oxygen demand (COD) of 4340 mg/L. Average digesting liquid temperatures ranged from 14.1 to 23.6 °C. At 6-day theoretical hydraulic residence times (V/Q where V is Lliquid, which is volume of liquid occupying the digester pores, and Q is total daily influent flow) and 1 reciprocation per day, methane production was 0.060 ± 0.10 LCH4/Lliquid-day and at 10 reciprocations methane production 0.058 ± 0.14 LCH4/Lliquid-day (mean ± standard deviation of measurements over time). COD percent removals were both 51% at 6-day V/Q. Since multiple reciprocations did not appear to make a difference in methane production and treatment performance, fewer reciprocations were used in subsequent experiments. Higher flow rates were also used in subsequent experiments to accelerate sludge clogging and channelization in the walnut-shell bed and thereby allow detection of any advantage provided by reciprocation compared to an upflow reactor. At 0 and 1 reciprocations per day and 0.35 and 0.50-day V/Qs, respectively, methane production was 0.24 ± 0.08 and 0.23 ± 0.08 LCH4/Lliquid-day and COD percent removal was 17 and 22%. Over the study period of 226 days, walnut shell porosities decreased due to sludge accumulation from 0.68 and 0.64 (start-up or clean-bed) to 0.31 and 0.24 in the 1 and 0 reciprocation per day reactors. Sludge accumulation and channelization did not appear to be affected by reciprocation mixing on the scale of this study.

Please read the abstract in the dissertation. Copyright / Dissertation (MEng)--University of Pretoria, 2011. / Chemical Engineering / unrestricted

A recombinat Chinese Hamster Ovary (rCHO) cell line designated as CHO SEAP was utilized in this investigation to optimize protein production. Two bench top stirred-tank bioreactors, namely a pitched-blade and a packed-bed basket bioreactor, were utilized for a comparative study to determine which bioreactor would produce the best results in terms of protein production. The objective of this research project was to provide basic data that shows cells cultured in a packed-bed basket bioreactor in perfusion mode will generate more protein product than cells in batch mode suspension culture with a pitched-blade bioreactor. The packed-bed bioreactor creates a homeostatic environment similar to the environment found in vivo, where waste products are constantly removed and fresh nutrients are replenished. Closed batch cultures do not provide a homeostatic environment. In batch culture systems, nutrients are depleted and waste products accumulate. The results from this experiment could help investigators involved in protein and/or vaccine production facilities select the appropriate bioreactor and mode of operation to optimize cell productivity for generation of a specific protein product. CHO cells have been used for the production of vaccines, recombinant therapeutic proteins, and monoclonal antibodies, and these cells are now the cell line of choice in the biopharmaceutical industry. Traditional vaccine production methods in egg embryos are slow and outdated, whereas roller bottle-based cell culture techniques are time consuming and have limited scalability. These limitations justify the need for development of stirred tank bioreactors. Cells cultured in a packed-bed bioreactor are not exposed to hydrodynamic forces, as is the case with pitched-blade bioreactors, allowing for maximum growth and protein expression. This mode of operation involves the constant removal of media depleted of nutrients and the addition of fresh media with more nutrients to keep the cells growing. Long run times decrease the constant need for re-seeding cells and re-establishing seed cultures, thus, reducing setup time and labor dramatically. Secreted products are automatically separated from cells in perfusion, eliminating filtration and membrane fouling. A detailed description of both modes of operation are discussed in this thesis.

No description available.