In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxygen transfer rate, volumetric and specific oxygen uptake rates, mass transfer and biochemical reaction resistances / moreover, the variation in product and by-product distribution, specific substrate uptake rates, yield and maintenance coefficient were investigated in the pilot scale batch bioreactor at QO/VR = 0.5 vvm and agitation rates of N= 250, 500, 625, and 750 min-1, and dissolved oxygen levels DO= 20%, 40% conditions, while medium components were CGlucose= 8.0 kg m-3, C(NH4)2HPO4= 5.0 kg m-3 and salt solution at controlled pHc=7.2. The highest cell concentration and benzaldehyde lyase activity were obtained at DO=40% condition as 3.0 kg m-3 and A=1095 Ucm-3, respectively. v Then a mathematical model was proposed to estimate benzaldehyde lyase activity as function of time, agitation rate, cell concentration, dissolved oxygen concentration, and by-product concentration with reasonable accuracy.

In this study, the effects of oxygen transfer (OT) on beta-lactamase production and on intracellular reaction rates were investigated with Bacillus licheniformis ATCC 2597. In order to clarify the oxygen transfer effects on the production of beta-lactamase, firstly a glucose based defined medium was designed and using this medium, the effects of bioreactor operation parameters, i.e., pH and temperature, on beta-lactamase activity and cell formation were investigated in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. Among the investigated bioprocess conditions, the highest beta-lactamase activity was obtained as A=115 U cm-3, in the medium with 7.0 kg m-3 glucose, 7.1 kg m-3 (NH4)2HPO4 and the salt solution, at pH0=7.5, T=37C, N=200 min-1. At the optimum conditions found in laboratory scale the effects of OT on cell generation, substrate consumption, product (beta-lactamase) and by-products formations were investigated at three different air inlet (Q0/ VR = 0.2, 0.5 and 1 vvm) and at three agitation rates (N=250, 500, 750 min-1) in V = 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Along with the fermentation, cell, substrate and by-product concentrations, beta-lactamase activity, yield coefficients, specific rates, oxygen uptake rates and the liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at 0.5 vvm 500 min-1 and at 0.2 vvm 500 min-1 conditions as ca. A=90 U cm-3 while the highest cell concentration was obtained as Cx=0.67 kg m-3 at 0.5 vvm 750 min-1 and at 0.2 vvm 750 min-1 conditions. KLa, increased with the increase in the agitation and aeration rates and its values varied between 0.007-0.044 s-1 and oxygen uptake rate varied between 0.4-1.6 mol m-3 s-1. Finally, the influence of OT conditions on the intracellular reaction rates was investigated using metabolic flux analysis to evaluate the effects of oxygen on the metabolism. Keywords: beta-lactamase, production, Bacillus, oxygen transfer, metabolic flux analysis

In this study, the effects of bioprocess operation parameters on benzaldehyde lyase production were systematically investigated. For this purpose, the research program was carried out in mainly four parts. In the first part of the study, Escherichia coli K12 (ATCC 10798), having the highest benzaldehyde lyase production capacity, was selected as the host microorganism. Next, using the selected microorganism, the production medium was designed in terms of its carbon and nitrogen sources. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 1.8 kg m-3 and 745 U cm-3, respectively, in the medium containing 8.0 kg m-3 glucose, 5.0 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, by using the designed medium, the effects of bioreactor operation parameters, i.e., oxygen transfer and pH, were investigated in pilot scale bioreactor. Oxygen transfer effects on benzaldehyde lyase production were investigated at QO/VR=0.5 vvm / N=250, 375, 500, 625, 750 min-1 and at QO/VR=0.7 vvm, N=750 min-1 conditions. The highest cell concentration and benzaldehyde lyase activity were obtained at 0.5 vvm, 500 min-1 condition as 2.3 kg m-3 and 860 U cm-3, respectively. Finally, the effect of pH was investigated for benzaldehyde lyase production process at Qo/VR=0.5 vvm, N=500 min-1 condition, at pHC=5.0, 6.4, 6.7, 7.0, 7.2 and 7.8 values. Among the investigated pH values, the highest cell concentration and enzyme activity were obtained at pHC=7.0 condition as 2.1 kg m-3 / 775 U cm-3. However, the values obtained at this condition, were lower than the values obtained at pHUC=7.2 uncontrolled pH operation. Hence, medium oxygen transfer condition and uncontrolled pH operation are found to be favorable for benzaldehyde lyase production.

In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3) pLySs cells, the effect of bioprocess parameters was systematically investigated. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 2.0 kg m-3 and 1060 U cm-3, respectively, in the medium containing 20.0 kg m-3 glucose, 11.8 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, oxygen transfer effects on benzaldehyde lyase production were investigated at air inlet v rate of QO/VR = 0.5 vvm, and agitation rates of N=500 and 750 min-1 and at QO/VR = 0.7 vvm, N=750 min-1 in pilot scale bioreactor and the highest cell concentration and volumetric BAL activity were found as 1.7 kg m-3 and 990 U cm-3, respectively, at 0.5 vvm, 750 min-1 condition. Next, the signal DNA sequence of serine alkaline protease (SAP) from B. licheniformis DSM 1969 chromosomal DNA (pre-subC) was fused in front of the bal by using PCR-based gene splicing by overlap extension (SOE) method. The fusion product of hybrid gene first cloned into pUC19 plasmid, thereafter sub-cloned into pBR374 shuttle vector and recombinant plasmid was transferred into various Bacillus species. However, no extracellular production of benzaldehyde lyase was observed in none of the developed recombinant Bacillus species, probably because of ineffective secretion system, inefficient folding of heterologous protein, degradation of enzyme due to proteolytic activity or high inactivation rate of the enzyme.

Made available in DSpace on 2014-06-11T19:29:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-28Bitstream added on 2014-06-13T18:38:59Z : No. of bitstreams: 1 araujo_ls_me_ilha.pdf: 2918640 bytes, checksum: 77955c3e7abab641549a8794e03fecfb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O reator aeróbio de leito fluidizado com circulação apresenta características que permitem efetuar o tratamento em apenas uma etapa. O uso deste reator torna-se atraente devido à possibilidade de mantê-lo com uma alta concentração de biomassa ativa, propiciando seu porte compacto, conseqüência do reduzido período de detenção hidráulica. A eficiência do biofilme no tratamento depende das condições hidrodinâmicas do reator, uma vez que sua espessura pode ser alterada de acordo com a velocidade de circulação, e a transferência de oxigênio através de suas camadas depende da vazão de ar injetada no sistema. O trabalho propôs-se a investigar a influência da relação entre diâmetros dos tubos concêntricos em quatro configurações, mantendo o diâmetro externo constante e igual a 250 mm e alterando o diâmetro interno entre 100, 125, 150 e 200 mm, proporcionando relações Ae/Ai iguais a 5,25; 3,00; 1,78 e; 0,56, respectivamente. O tubo interno possui 2,22 m e é posicionado 0,05m acima do fundo do reator. A altura estática da coluna de fluido foi 2,35 m. Os ensaios hidrodinâmicos – velocidade do líquido, fração volumétrica de ar, tempo de mistura – e os de transferência de oxigênio foram realizados para vazões de ar até 2500 l/h em meio bifásico e em meio trifásico com concentrações de areia iguais a 30, 70, 100 e 150 g/l. O diâmetro interno de 125 mm foi o mais eficiente na indução de velocidade de circulação do líquido. A fração volumétrica de ar aumentou com o aumento do diâmetro interno para mesmas velocidades superficiais de ar, com exceção do menor diâmetro, o qual apresentou comportamento diferenciado dos demais. Os reator com diâmetro interno de 150 mm apresentou maiores valores de KLa para as mesmas vazões de ar. Observou-se que a adição de sólidos causou um decréscimo da velocidade do líquido e da fração... / Aerobic internal-loop airlift reactor has some characteristics that allow wastewater treatment in a single stage. The attractive to use this kind of reactor is mainly because of its capacity of maintaining a high active biomass concentration into it, which induces its small structure, in a consequence of its small hydraulic detention time. The biofilm efficiency in the treatment depends on reactor’s hydrodynamic conditions. The structure of the biofilm and the oxygen transfer into its layers is affected by the liquid velocity induced by the air flow rate. This work proposed an investigation about the influence of the diameter of the concentric tubes, using four geometric configurations. The model had a 250 mm external tube and the inner draft tube with diameters of 100, 125, 150 and 200 mm, which means AD/AR equals to 5.25, 3.00, 1.78 and 0.56, respectively. The draft tube’s height was 2,22 m and was positioned 0,05 m above the bottom of the reactor. The static fluid height was 2,35 m. The hydrodynamic tests – liquid velocities, gas hold up, mixture time – and the oxygen transfer tests were conducted for two and three-phase systems, with variable air flow rate up to 2500 l/h. The solid phase concentrations used were 30, 70, 100 and 150 g/l. The 125 mm reactor was the most efficient in inducing the liquid circulation velocity. The gas hold up increased with the increasing of the draft tube diameter, for same superficial gas velocities, excepting the 100 mm, which showed a different behavior. The 150 mm reactor presented the highest values for KLa for the same air rate. It was noticed that the solids addiction decreased the liquid velocity, gas hold up and oxygen transfer. Hydrodynamics equations found in literature showed a limited applicability, evidencing the necessity of relations capable to reach the most different configuration for this kind of reactor. A mass transfer mathematic...(Complete abstract click electronic acces below)

Orientador: Maria Helena Andrade Santana / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-09T16:06:29Z (GMT). No. of bitstreams: 1 Bastos_ReinaldoGaspar_D.pdf: 5701398 bytes, checksum: ea21a329b956587a06c56820ffbe5b4e (MD5) Previous issue date: 2006 / Resumo: O cultivo em estado sólido tem merecido expressiva atenção nos últimos anos pelas vantagens oferecidas principalmente nos processos que envolvem fungos filamentosos. Entretanto, o interesse pelos fenômenos de transferência de massa e pelo desempenho de biorreatores é objeto de estudos mais recentes, motivado pelo escalonamento dos processos. Nesse contexto, o presente trabalho tem como objetivo estudar a transferência de oxigênio no processo de produção de proteínas alergênicas obtidas da biomassa de Drechslera (Helminthosporium) monoceras por cultivo em estado sólido em colunas de leito fixo com farelo de trigo e espuma de poliuretano. Os resultados indicam que vazões de ar da ordem de 1L/min e 30mm de altura de leito de partículas mantêm a umidade inicial do meio sólido praticamente constante para farelo de trigo. Essas condições asseguram os melhores valores de KLas, além de minimizar a compactação do leito, permitir o crescimento na fase aérea e assegurar a ausência de água livre durante o processo. O cultivo em espuma de poliuretano apresenta produção de proteínas superior aos ensaios com farelo de trigo devido à manutenção das propriedades físicas da matriz sólida inerte. Foram identificadas proteínas na faixa de 15 a 139kDa para ensaios em farelo de trigo e 11 a 244kDa sobre espuma de poliuretano, sendo que as frações indicadas como mais alergênicas 14,4, 36 e 60kDa foram encontradas para os dois tipos de suportes. Estes resultados mostram que condições selecionadas de vazão de ar, altura de leito e suporte sólido preservam a ausência de água livre no cultivo em estado sólido, aumentando a produção das proteínas alergênicas e facilitando o controle do processo / Abstract: In recent years, a resurgence of interest in solid-state cultivation has been observed due to its numerous advantages, mainly concerning the growth of filamentous fungi. However, studies on the oxygen transfer and performance of bioreactors are still scarce. The main difficulties faced by process scale-up are the control of parameters and limiting oxygen transfer. The aim of thisresearch was to study the oxygen transfer for the production of allergenic proteins from the biomass of Drechslera (Helminthosporium) monoceras cultured on wheat bran and polyurethane foam. Results indicated that flow rates about 1L/min and 30mm of bed height lead to the best values of overall oxygen transfer coefficient (KLa) on wheat bran, showing no substrate bed packing and formation of aerial hyphae, maintaining the characteristics of solid state cultivation. Evaluation of polyurethane foam showed high protein production, without changing the physical properties of support. SDS-Page assays identified proteins from crude extract in an approximate range of 15 to 139kDa and 11to 244kDa for polyurethane foam and for wheat bran, respectively. The pool of proteins obtained includes the molecular masses, identified in previous works, as the most allergenic fractions (14,4, 36 and 60kDa) These results show that selected conditions of air-flow rate, bed height and solid support preserve the absence of free water in solid state cultivation, increasing the production of allergenic proteins and facilitating the control of the process / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Orientador: Milton Dall'Aglio Sobrinho / Banca: Tsunao Matsumoto / Banca: Gilson Arimura Arima / Resumo: O reator aeróbio de leito fluidizado com circulação apresenta características que permitem efetuar o tratamento em apenas uma etapa. O uso deste reator torna-se atraente devido à possibilidade de mantê-lo com uma alta concentração de biomassa ativa, propiciando seu porte compacto, conseqüência do reduzido período de detenção hidráulica. A eficiência do biofilme no tratamento depende das condições hidrodinâmicas do reator, uma vez que sua espessura pode ser alterada de acordo com a velocidade de circulação, e a transferência de oxigênio através de suas camadas depende da vazão de ar injetada no sistema. O trabalho propôs-se a investigar a influência da relação entre diâmetros dos tubos concêntricos em quatro configurações, mantendo o diâmetro externo constante e igual a 250 mm e alterando o diâmetro interno entre 100, 125, 150 e 200 mm, proporcionando relações Ae/Ai iguais a 5,25; 3,00; 1,78 e; 0,56, respectivamente. O tubo interno possui 2,22 m e é posicionado 0,05m acima do fundo do reator. A altura estática da coluna de fluido foi 2,35 m. Os ensaios hidrodinâmicos - velocidade do líquido, fração volumétrica de ar, tempo de mistura - e os de transferência de oxigênio foram realizados para vazões de ar até 2500 l/h em meio bifásico e em meio trifásico com concentrações de areia iguais a 30, 70, 100 e 150 g/l. O diâmetro interno de 125 mm foi o mais eficiente na indução de velocidade de circulação do líquido. A fração volumétrica de ar aumentou com o aumento do diâmetro interno para mesmas velocidades superficiais de ar, com exceção do menor diâmetro, o qual apresentou comportamento diferenciado dos demais. Os reator com diâmetro interno de 150 mm apresentou maiores valores de KLa para as mesmas vazões de ar. Observou-se que a adição de sólidos causou um decréscimo da velocidade do líquido e da fração... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aerobic internal-loop airlift reactor has some characteristics that allow wastewater treatment in a single stage. The attractive to use this kind of reactor is mainly because of its capacity of maintaining a high active biomass concentration into it, which induces its small structure, in a consequence of its small hydraulic detention time. The biofilm efficiency in the treatment depends on reactor's hydrodynamic conditions. The structure of the biofilm and the oxygen transfer into its layers is affected by the liquid velocity induced by the air flow rate. This work proposed an investigation about the influence of the diameter of the concentric tubes, using four geometric configurations. The model had a 250 mm external tube and the inner draft tube with diameters of 100, 125, 150 and 200 mm, which means AD/AR equals to 5.25, 3.00, 1.78 and 0.56, respectively. The draft tube's height was 2,22 m and was positioned 0,05 m above the bottom of the reactor. The static fluid height was 2,35 m. The hydrodynamic tests - liquid velocities, gas hold up, mixture time - and the oxygen transfer tests were conducted for two and three-phase systems, with variable air flow rate up to 2500 l/h. The solid phase concentrations used were 30, 70, 100 and 150 g/l. The 125 mm reactor was the most efficient in inducing the liquid circulation velocity. The gas hold up increased with the increasing of the draft tube diameter, for same superficial gas velocities, excepting the 100 mm, which showed a different behavior. The 150 mm reactor presented the highest values for KLa for the same air rate. It was noticed that the solids addiction decreased the liquid velocity, gas hold up and oxygen transfer. Hydrodynamics equations found in literature showed a limited applicability, evidencing the necessity of relations capable to reach the most different configuration for this kind of reactor. A mass transfer mathematic...(Complete abstract click electronic acces below) / Mestre

Seawater flue gas desulphurization (SWFGD) is a reliable and efficient process to re-move sulfur dioxide in flue gases in power plants and industries. The characteristics and applications of seawater treatment plant of three SWFGD process are described firstly. A literatual survey looking at different air injection techniques in shallow water basin applications are investigated, also including best practice from similar applications in other types of industries. A number of promising alternatives is then selected and compared with today’s system. The relevant experimental data of these systems are collected. A rate-based model is developed to simulate and optimize the aeration process in seawater treatment plant after its reliability is verified by the experimental data.

Seawater flue gas desulphurization (SWFGD) is a reliable and efficient process to re-move sulfur dioxide in flue gases in power plants and industries. The characteristics and applications of seawater treatment plant of three SWFGD process are described firstly. A literatual survey looking at different air injection techniques in shallow water basin applications are investigated, also including best practice from similar applica-tions in other types of industries. A number of promising alternatives is then selected and compared with today’s system. The relevant experimental data of these systems are collected. A rate-based model is developed to simulate and optimize the aeration process in seawater treatment plant after its reliability is verified by the experimental data.

A submerged-media biological aerated filter (BAF) has been studied to 1) evaluate oxygen transfer kinetics under conditions without biological growth and 2) determine the influence of biological growth on the rate of oxygen transfer. Collectively, the study evaluates the rates of supply and consumption of oxygen in BAFs. The mass-transfer characteristics of a submerged-media BAF were initially determined over a wide range of gas and liquid flow rates without the presence of bacteria. The mass-transfer coefficients (KLa(T)) were measured using a nitrogen gas stripping method and were found to increase as both gas and liquid superficial velocities increase, with values ranging from approximately 40 to 380 h??. The effect of parameters including the gas and liquid velocities, dirty water to clean water ratio, and temperature dependence was successfully correlated within +/- 20% of the experimental KLa value. The effects of the media size and gas holdup fractions were also investigated. Stagnant gas holdup did not significantly influence the rate of oxygen transfer. Dynamic gas holdup and the difference between total and stagnant gas holdup were found to increase with an increase in gas velocity. Neither liquid velocity nor liquid temperature was determined to have a significant impact on gas holdup. A tertiary nitrification BAF pilot unit was then operated for 5 months downstream of a secondary treatment unit at a domestic wastewater treatment facility. The study investigated the oxygen transfer capabilities of the nitrifying unit with high oxygen demand requirements through a series of aeration process tests and explored the presence of oxygen transfer enhancements by further analyzing the actual transfer mechanism limitations. It was determined that (assuming OTE equals 20 percent) aerating the BAF pilot unit based on the stoichiometric aeration demand resulted in overaeration of the unit, especially at lower pollutant loading rates. Endogenous respiration contributed to only 2 to 7 percent of the total oxygen demand with regions of biomass activity changing with varying loading conditions. An enhanced oxygen transfer factor was determined in the biologically active pilot. Although it cannot be definitively concluded that the observed oxygen transfer factor is either due to biological activity or not simply an artifact of measurement/analysis techniques, the enhancement factor can be mathematically accounted for by either an increase in the KLa factor or the associated driving force using a proposed enhanced bubble theory. / Master of Science