The aeration potential of a low profile cascade aerator was studied under varying operational conditions in accordance with the ASCE Standard for Measurement of Oxygen Transfer in Clean Water [ASCE 2-06, 2007]. Operational parameters delved into included the channel slope (2.50, 4.50 and 6.50); water flow rate (465.75 L/min.m (37.5 gpm/ft), 931.45 L/min.m (75 gpm/ft) and 1397.20 L/min.m (112.5 gpm/ft)); and weir geometry (rectangular-shaped, inverted T-shaped, W-shaped and inverted Cross shaped weir). The oxygen transfer coefficient, KLa, was derived by use of a FORTRAN-based nonlinear regression analysis computer program and served to assess the effectiveness of various combinations of operational parameters. Statistical tests (ANOVA analysis and main plot, interactive plot) were performed on the results to determine the optimal operating conditions. It was discovered that the combination of the inverted Cross shaped weir and flow rates of 1397.20 L/min.m (112.5 gpm/ft) produced the highest reaeration rates for all slope considered. On the other hand, the W-shaped weir produced better reaeration values at lower flows of 465.75 L/min.m (37.5 gpm/ft) and 931.45 L/min.m (75 gpm/ft) for the range of channel slopes examined. These effects can be respectively attributed to the strong turbulent mixing generated by the plunging nappe flow and recirculating air vortices, which apparently led to substantial air entrainment in the water mass.

A ocorrência da redução biológica do sulfato resulta na formação de um produto indesejado, o sulfeto de hidrogênio, que pode ser removido sob condições limitadas de oxigênio. Os fatores que podem afetar a oxidação do sulfeto não estão bem elucidados e o processo de microaeração é pouco utilizado devido a limitações tecnológicas. Desta forma a oxidação química e bioquímica do sulfeto foi estudada em duas configurações de reatores que utilizam membrana polimérica porosa (membrana de silicone) como alternativa para microaeração. As configurações apresentam duas formas de microaeração, por pressão interna (Reator de membrana Submersa-RMS) e outra externa (Reator de Membrana Externa-RME) com aeração por troca atmosférica. Foram realizados ensaios de transferência de massa para determinação do coeficiente volumétrico global de transferência (KLa). Os resultados médios obtidos para o KLa foram de 0,10 h-1 para o RMS e 0,154 h-1 para o RME, confirmando a microaeração do meio. Os reatores foram operados a fim de avaliar a oxidação do sulfeto a enxofre elementar por via química e bioquímica em diferentes faixas de pH, pressão (para o RMS) e velocidade no interior da membrana (para o RME) por meio de planejamento fatorial. A oxidação química apresentou alta conversão do sulfeto a enxofre com percentual médio de 91,3% no RMS e 79,2% no RME e percentual médio de conversão a sulfato de 2,3% no RMS e 15,7% no RME. A oxidação bioquímica no RME apresentou valores médios de conversão de sulfeto a enxofre de 41,6% e de 52,8% para a conversão a sulfato. A oxidação de efluente de reator sulfetogênico também foi avaliada atingiu valores médios de 48,7% para a conversão a enxofre elementar 46,7% para a conversão de sulfeto a sulfato. O estudo das variáveis demonstrou que a variável que influenciou a oxidação foi o pH. A configuraçõa RMS apresentou problemas operacionais inviabilizando seu uso. O uso de membranas apresentou viabilidade de controle da microaeração através das variáveis operacionais, raio e espessura da membrana. Desta forma, devido à viabilidade operacional e os resultados obtidos, as configurações se mostraram uma alternativa viável a conversão do sulfeto a enxofre elementar. / The occurrence of biological sulfate reduction results in the formation of unwanted hydrogen sulfide, which can be removal under oxygen-limiting conditions. Detailed knowledge of this process remains unclear and the micro aeration process was prevented by technological limitations. For these reasons, the chemical and biological oxidation of sulfide was studied in two configurations of reactors using porous polymer membrane (silicon membrane) as an alternative to micro aeration. The configurations present two forms of micro-aeration by internal pressure (Submerged Membrane Reactor- SMR) and an external one (External Membrane Reactor EMR) with aeration by air exchange. The micro aeration was evaluated through the global oxygen mass transfer coefficient (KLa). The average results obtained for KLa were 0.10 h-1 for SMR and 0.154 h-1 for EMR, confirming the micro aeration. The settings were operated to evaluate the chemical and biochemical oxidation of sulfide to elemental sulfur by different variables: pH, pressure (for SMR) and flow/membrane area (for EMR) through factorial design. The chemical oxidation showed high sulfide conversion to sulfur, with mean value of 91.3% in SMR and 79.2% in EMR and conversion to sulfate of 2.3% in SMR and 15.7% in EMR. The biochemical oxidation in EMR showed mean values of sulfide conversion to sulfur of 41.6% and 52.8% for sulfate. The oxidation of sulfidogenic reactor effluent was evaluated and obtained mean values of 48.7% for conversion to elemental sulfur and 46.7% to sulfate. The variable studies showed that the variable pH is significantly for sulfide oxidation. The SMR presented operational problems invalidating their use. The use of membranes, showed a high viability due to its ability to micro aeration especially because of the operating variables, radius and membrane thickness. In general the reactors were efficient due the low sulfate yields. Thus the reactors proved are viable settings to the conversion of sulfide to elemental sulfur.

A ocorrência da redução biológica do sulfato resulta na formação de um produto indesejado, o sulfeto de hidrogênio, que pode ser removido sob condições limitadas de oxigênio. Os fatores que podem afetar a oxidação do sulfeto não estão bem elucidados e o processo de microaeração é pouco utilizado devido a limitações tecnológicas. Desta forma a oxidação química e bioquímica do sulfeto foi estudada em duas configurações de reatores que utilizam membrana polimérica porosa (membrana de silicone) como alternativa para microaeração. As configurações apresentam duas formas de microaeração, por pressão interna (Reator de membrana Submersa-RMS) e outra externa (Reator de Membrana Externa-RME) com aeração por troca atmosférica. Foram realizados ensaios de transferência de massa para determinação do coeficiente volumétrico global de transferência (KLa). Os resultados médios obtidos para o KLa foram de 0,10 h-1 para o RMS e 0,154 h-1 para o RME, confirmando a microaeração do meio. Os reatores foram operados a fim de avaliar a oxidação do sulfeto a enxofre elementar por via química e bioquímica em diferentes faixas de pH, pressão (para o RMS) e velocidade no interior da membrana (para o RME) por meio de planejamento fatorial. A oxidação química apresentou alta conversão do sulfeto a enxofre com percentual médio de 91,3% no RMS e 79,2% no RME e percentual médio de conversão a sulfato de 2,3% no RMS e 15,7% no RME. A oxidação bioquímica no RME apresentou valores médios de conversão de sulfeto a enxofre de 41,6% e de 52,8% para a conversão a sulfato. A oxidação de efluente de reator sulfetogênico também foi avaliada atingiu valores médios de 48,7% para a conversão a enxofre elementar 46,7% para a conversão de sulfeto a sulfato. O estudo das variáveis demonstrou que a variável que influenciou a oxidação foi o pH. A configuraçõa RMS apresentou problemas operacionais inviabilizando seu uso. O uso de membranas apresentou viabilidade de controle da microaeração através das variáveis operacionais, raio e espessura da membrana. Desta forma, devido à viabilidade operacional e os resultados obtidos, as configurações se mostraram uma alternativa viável a conversão do sulfeto a enxofre elementar. / The occurrence of biological sulfate reduction results in the formation of unwanted hydrogen sulfide, which can be removal under oxygen-limiting conditions. Detailed knowledge of this process remains unclear and the micro aeration process was prevented by technological limitations. For these reasons, the chemical and biological oxidation of sulfide was studied in two configurations of reactors using porous polymer membrane (silicon membrane) as an alternative to micro aeration. The configurations present two forms of micro-aeration by internal pressure (Submerged Membrane Reactor- SMR) and an external one (External Membrane Reactor EMR) with aeration by air exchange. The micro aeration was evaluated through the global oxygen mass transfer coefficient (KLa). The average results obtained for KLa were 0.10 h-1 for SMR and 0.154 h-1 for EMR, confirming the micro aeration. The settings were operated to evaluate the chemical and biochemical oxidation of sulfide to elemental sulfur by different variables: pH, pressure (for SMR) and flow/membrane area (for EMR) through factorial design. The chemical oxidation showed high sulfide conversion to sulfur, with mean value of 91.3% in SMR and 79.2% in EMR and conversion to sulfate of 2.3% in SMR and 15.7% in EMR. The biochemical oxidation in EMR showed mean values of sulfide conversion to sulfur of 41.6% and 52.8% for sulfate. The oxidation of sulfidogenic reactor effluent was evaluated and obtained mean values of 48.7% for conversion to elemental sulfur and 46.7% to sulfate. The variable studies showed that the variable pH is significantly for sulfide oxidation. The SMR presented operational problems invalidating their use. The use of membranes, showed a high viability due to its ability to micro aeration especially because of the operating variables, radius and membrane thickness. In general the reactors were efficient due the low sulfate yields. Thus the reactors proved are viable settings to the conversion of sulfide to elemental sulfur.

På Käppalaverket i Stockholm står luftningen av de biologiska bassängerna för omkring en femtedel av verkets totala elenergiförbrukning. I ett försök att minska energikostnaden utvärderades under hösten 2010 nya metoder för luftflödesreglering på verket. Grundtanken var att styra luftflödet efter medelvärdet på utgående ammoniumkoncentration under en längre tid, istället för som idag efter momentana värden. Ett vanligt sätt att styra luftflöden på reningsverk idag är att använda återkoppling från utgående ammoniumkoncentration, vilket syftar till att alltid hålla den utgående koncentrationen vid ett valt börvärde. Lagstiftade gränsvärden på ammonium avser dock normalt medelvärden över en längre tid, såsom kvartal eller år. Istället för att anpassa luftflödet efter den inkommande belastningen är det därför möjligt att hålla luftflödet relativt konstant medan istället den utgående koncentrationen tillåts variera. I denna studie visades en energibesparing kunna erhållas om luftflödets variation reduceras. Två strategier utvärderades i vilka luftflödet respektive syrehalten hölls så konstant som möjligt. Dessa jämfördes med den idag använda styrstrategin på Käppalaverket, i vilken luftflödet anpassas efter den inkommande belastningen genom återkoppling. Studien inkluderade både simuleringar i modellen Benchmark Simulation Model no. 1 och fullskaleförsök på Käppalaverket. I både simuleringar och fullskaleförsök resulterade de två utvärderade strategierna i en lägre luftförbrukning per reningsgrad än den idag använda återkopplingsstrategin. I fullskaleförsöken erhölls en luftflödesreduktion på 11 % då luftflödet hölls konstant och 15 % då syrehalten hölls konstant. Båda strategierna genererade dock en kraftigt varierande utgående ammoniumkoncentration. Variationerna var störst då luftflödet hölls konstant och korrelerade inte med den dygnsbaserade belastningsprofilen. Sammanfattningsvis visade studien att en reducering av luftflödets variation resulterar i en lägre luftförbrukning men också i en ökad instabilitet. En konstant syrehalt gav en större energivinst och även en stabilare ammoniumreduktion än ett konstant luftflöde, varför denna metod har störst potential till vidare implementering i fullskala. / The aeration of the bioreactors is responsible for one fifth of the energy consumption at the Käppala wastewater treatment plant (WWTP) in Stockholm. In this report, new methods for aeration control were evaluated in order to reduce the energy costs at the plant. The main idea was to control the effluent ammonia concentration in terms of mean values instead of momentary values. A quite common approach for aeration control is to use feedback from the effluent ammonia concentration, thus aiming to keep the effluent concentration consistently at a certain set point. However, discharge limits normally refer to mean values over longer periods of time, such as months or years. Instead of adjusting the airflow to the incoming load it is therefore possible the keep the airflow fairly constant while allowing a fluctuating effluent concentration. In this paper, it was shown that by reducing the variation of the airflow, energy could be saved. Two methods were evaluated in which the airflow and oxygen concentration respectively was held constant. These methods were compared to the control strategy used today at the Käppala WWTP, where feedback control adjusts the airflow to the influent load. The study consisted of simulations with the Benchmark simulation model no. 1 (BSM1) as well as full scale experiments at the Käppala WWTP. Both the simulations and full scale experiments showed a reduced aeration per nutrient removal for the evaluated methods. In full scale, the total airflow reduction was 11 % when the airflow was held constant and 15 % when the oxygen concentration was held constant. However, the methods resulted in large variations of the effluent ammonia concentration, which did not correlate to the daily influent load. The variations were especially large when the airflow was held constant. In summary, this study showed that a reduced airflow variation results in lower aeration costs but also less stability. A constant oxygen concentration required less aeration and provided a more stable degree of ammonia removal than a constant airflow. For this reason, aeration control with a constant oxygen concentration has the best potential for further use at the Käppala WWTP.

O fragmento de anticorpo scFv foi expresso pela levedura Pichia pastoris recombinante utilizando o promotor AOX1, em presença de metanol, como fonte de carbono e como modelo de expressão. Este anticorpo é considerado uma possível alternativa para o diagnóstico e tratamento da aterosclerose. Foi realizado um estudo inicial da produção do fragmento de anticorpo e do crescimento da levedura com dois bancos de trabalho diferentes, variando a fonte de carbono (glicose e glicerol). Os resultados obtidos demonstraram que as máximas concentrações celulares e a maior produção de anticorpo foram atingidas com o estoque de trabalho mantido com glicerol como fonte de carbono e de preservação. Posteriormente, foi realizado um estudo em shaker e biorreator a partir de um planejamento fatorial completo 23 em que se estudaram as variáveis tempo de indução (24, 28 e 32 horas), temperatura de indução (10, 20 e 30°C), e concentração de indutor (metanol 0,5, 1,0 e 1,5%) sobre a variável resposta produção do fragmento de anticorpo. A única variável que apresentou efeito estatisticamente significativo, com nível de confiança de 95%, foi a concentração de indutor para os dois sistemas de produção. As melhores produções de fragmento de anticorpo foram de 27,8 mg/L em shaker e de 41,3 mg/L em biorreactor. O terceiro estudo consistiu em avaliar a produção do fragmento de anticorpo em shaker pela levedura em distintas fontes de carbono (sacarose, glicerol, glicose e sacarose/glicose 50%). Neste estudo a analise de ANOVA não apresentou diferenças estatísticas significativas. Foi realizado um estudo para melhorar a quantificação do fragmento de anticorpo visando diminuir o número de etapas para evitar perdas no processo. Após obter um novo protocolo para a quantificação, realizou-se um novo estudo para avaliar a produção com as diferentes fontes de carbono. Com a analise dos resultados observou-se que todas as fontes de carbono apresentaram efeitos estatisticamente significativos, sendo que com a sacarose se obteve a maior produção com 93,7 mg/L do fragmento de anticorpo. Estes resultados foram comparados e analisados com observações realizadas ao microscópio da levedura, que demonstraram que a melhor produção de anticorpo foi obtida no momento que a levedura apresentava diâmetros menores que 0,895 µm. Seguidamente foi realizado um estudo para determinar fontes alternativas de carbono e nitrogênio (farelo de arroz e farelo de trigo) para produzir o fragmento de anticorpo, em substituição de subtratos como peptona de caseína e casaminoacidos, a maior produção obtida neste caso foi de 27,9 mg/L quando foi trocado o casaminoacidos pelo farelo de trigo. Finalmente foi avaliado o efeito do kLa no crescimento da levedura e a produção do fragmento de anticorpo scFv, os resultados demonstraram que a melhor condição para o crescimento da levedura foi com kLa 96,12 h-1, por outro lado a melhor condição para a produção do fragmento de anticorpo foi de 12,24 h-1 com relação a produção final de 8,12 mg/L de anticorpo/ g/L de célula. / The scFv antibody fragment was expressed in the recombinant yeast Pichia pastoris by using AOX1 promoter, in the presence of methanol, as carbon source and as a model for expression. This antibody is considered as a possible alternative for the diagnosis and treatment of atherosclerosis. An initial study was conducted in the production of the antibody fragment and the growth of yeast with two different work benches, varying the carbon source (glucose and glycerol). The results showed that the maximum cell concentrations, and the best production of antibody, were achieved with maintained working stock with glycerol as carbon source and preservation. Subsequently, a study was conducted in shaker and bioreactor, from a 23 complete factorial design, in which the variables studied were time induction (24, 28 and 32 hours), temperature induction (10, 20 and 30°C), and concentration of the inducer (methanol 0.5, 1.0 and 1.5%) on the response variable production of the antibody fragment. The only variable that had a statistically significant effect, with a confidence level of 95%, was the inducer concentration for the two production systems. The best yields of the antibody fragment were of 27.8 mg/L for the shaker and 41.3 mg/L for the bioreactor. The third study was to assess the production of antibody fragment into the shaker with the yeast at different carbon sources (sucrose, glycerol, glucose and sucrose/glucose 50%). In this study, the ANOVA analysis did not show statistically significant differences. We conducted planning to improve the quantification of antibody fragment in order to decrease the number of steps to avoid losses in the process. After getting a new protocol for the quantification, we carried out a new study to evaluate the production with different carbon sources. The analysis of the results showed that all carbon sources showed statistically significant effects, among which sucrose obtained the highest yield 93.7 mg/L of antibody fragment. These results were compared and analyzed through a microscope, in which with the observations of the yeast showed that the best antibody production was obtained when the yeast had sizes smaller than 0.895 microns. Next, a study was conducted to determine alternative sources of carbon and nitrogen (rice bran and wheat bran) to produce the antibody fragment in place of casein peptone and casaminoacids as substrates. The highest yield obtained in this case was 27.9 mg/L, when casamino was replaced by the wheat bran. Finally, we evaluated the effect of kLa in yeast growth and production of the antibody fragment scFv, the results showed that the best condition for the growth of the yeast was 96.12 h-1, on the other hand, the best condition for the production antibody fragment was 12.24 h-1 with a final relation yield of 8.12 mg/L antibody/g/L cell.

Limitierend für pharmakologische Therapien ist oft die Unfähigkeit des Wirkstoffes, biologische Membranen zu überwinden, weswegen häufig Transportmoleküle wie z.B. zellpenetrierende Peptide (CPPs, cell penetrating peptides) benutzt werden. Von den über 100 beschriebenen CPPs wurde bisher nur eine kleine Anzahl systematisch verglichen, was die Auswahl des „richtigen“ CPPs für eine Anwendung erschwert. Ziel dieser vorliegenden Arbeit war es, das pro-apoptotische Peptid KLA mittels CPPs spezifisch in Krebszellen zu transportieren. Untersucht wurden: (I) Verschiedene CPPs in unterschiedlichen Zelltypen zur Selektion der „besten“ CPPs; (II) Der Einflusses des CPP C-Terminus auf die Internalisierung und zelluläre Verteilung; (III) Der zelluläre KLA-Transport mittels CPPs via einer nicht-kovalenten Administration. 22 verschiedene CPPs wurden in sieben Zelltypen untersucht, wobei Toxizität, Zellaufnahme und zelluläre Lokalisation mittels Fluorescein markierten CPPs in Fluoreszenzspektroskopie und konfokaler Mikroskopie betrachtet wurden. Abhängig von der Zellaufnahme wurden die CPPs in drei Gruppen klassifiziert. Die Untersuchung carboxylierter und carboxyamidierter CPP C-Termini ergab, dass in den meisten Fällen ein Carboxyamid die zelluläre Aufnahme begünstigte. Drei CPPs (MPG, Penetratin und Integrin) wurden ausgewählt, um das pro-apoptotische KLA Peptid in zwei Krebszelllinien (MCF-7 Brustkrebszellen und leukämische RAW264.7 Makrophagen) im Vergleich zu Fibroblasten (Cos-7) nicht-kovalent zu transportieren. Der erfolgreiche KLA-Transport hing vom CPP, dessen C-Terminus und der Zelllinie ab. Die Analyse der Viabilität nach CPP:KLA Administration ergab, dass MPG-CONH2:KLA (1:2) toxisch für Makrophagen und Brustkrebszellen, aber nicht für Fibroblasten war. Die Toxizität konnte der Apoptose zugeordnet werden. Die vorliegende Arbeit liefert wichtige Informationen über die Auswahl des passenden CPPs für den nicht-kovalenten Transport des pro-apoptotischen KLA-Peptids. / Limitations in a pharmacological therapy are often due to the inability of drugs to overcome the cell membrane and therefore transporting molecules are being used, e.g. cell penetrating peptides (CPPs). Only a few of the over 100 described CPPs have been compared systematically making the choice of “the” CPP for a given application difficult. The goal of the presented work is the CPP mediated delivery of the pro-apoptotic peptide KLA in breast cancer cells as proof of principle for a therapeutical application. Analysed were (I) Different CPPs in various cell types to select the “best” one, (II) The influence of the CPP C-termini on uptake and localisation, (III) The cellular KLA delivery via a non-covalent CPP administration. 22 CPPs were compared in seven cell types thereby looking at toxicity, cellular uptake and subcellular localisation using fluorescein labelled CPPs for fluorescence spectroscopy and confocal microscopy. The resulting uptake information allowed the classification of the CPPs in three main groups. The evaluation of carboxylated and carboxyamidated CPP C-termini revealed that a carboxyamide mostly enhanced the cellular CPP uptake. Three CPPs were selected (MPG, penetratin and integrin) to deliver the pro-apoptotic KLA peptide in two cancer cell lines (breast cancer MCF-7 cells and RAW264.7 macrophages) compared to fibroblasts (Cos-7) via the non-covalent strategy. A successful KLA delivery depended on the applied CPP, its C-terminus and the used cell line. The biological activity of the pro-apoptotic KLA peptide was determined via the cell viability (MTT assay). The co-incubation of MPG-CONH2:KLA (1:2) was able to induce toxicity in breast cancer cells and leukaemic macrophages, but not in fibroblasts. The viability reductions were then assigned to apoptosis. This work provides important information for the choice of an adequate CPP for the pro-apoptotic KLA peptide delivery and presents the advantage of the non-covalent delivery strategy.

O fragmento de anticorpo scFv foi expresso pela levedura Pichia pastoris recombinante utilizando o promotor AOX1, em presença de metanol, como fonte de carbono e como modelo de expressão. Este anticorpo é considerado uma possível alternativa para o diagnóstico e tratamento da aterosclerose. Foi realizado um estudo inicial da produção do fragmento de anticorpo e do crescimento da levedura com dois bancos de trabalho diferentes, variando a fonte de carbono (glicose e glicerol). Os resultados obtidos demonstraram que as máximas concentrações celulares e a maior produção de anticorpo foram atingidas com o estoque de trabalho mantido com glicerol como fonte de carbono e de preservação. Posteriormente, foi realizado um estudo em shaker e biorreator a partir de um planejamento fatorial completo 23 em que se estudaram as variáveis tempo de indução (24, 28 e 32 horas), temperatura de indução (10, 20 e 30°C), e concentração de indutor (metanol 0,5, 1,0 e 1,5%) sobre a variável resposta produção do fragmento de anticorpo. A única variável que apresentou efeito estatisticamente significativo, com nível de confiança de 95%, foi a concentração de indutor para os dois sistemas de produção. As melhores produções de fragmento de anticorpo foram de 27,8 mg/L em shaker e de 41,3 mg/L em biorreactor. O terceiro estudo consistiu em avaliar a produção do fragmento de anticorpo em shaker pela levedura em distintas fontes de carbono (sacarose, glicerol, glicose e sacarose/glicose 50%). Neste estudo a analise de ANOVA não apresentou diferenças estatísticas significativas. Foi realizado um estudo para melhorar a quantificação do fragmento de anticorpo visando diminuir o número de etapas para evitar perdas no processo. Após obter um novo protocolo para a quantificação, realizou-se um novo estudo para avaliar a produção com as diferentes fontes de carbono. Com a analise dos resultados observou-se que todas as fontes de carbono apresentaram efeitos estatisticamente significativos, sendo que com a sacarose se obteve a maior produção com 93,7 mg/L do fragmento de anticorpo. Estes resultados foram comparados e analisados com observações realizadas ao microscópio da levedura, que demonstraram que a melhor produção de anticorpo foi obtida no momento que a levedura apresentava diâmetros menores que 0,895 µm. Seguidamente foi realizado um estudo para determinar fontes alternativas de carbono e nitrogênio (farelo de arroz e farelo de trigo) para produzir o fragmento de anticorpo, em substituição de subtratos como peptona de caseína e casaminoacidos, a maior produção obtida neste caso foi de 27,9 mg/L quando foi trocado o casaminoacidos pelo farelo de trigo. Finalmente foi avaliado o efeito do kLa no crescimento da levedura e a produção do fragmento de anticorpo scFv, os resultados demonstraram que a melhor condição para o crescimento da levedura foi com kLa 96,12 h-1, por outro lado a melhor condição para a produção do fragmento de anticorpo foi de 12,24 h-1 com relação a produção final de 8,12 mg/L de anticorpo/ g/L de célula. / The scFv antibody fragment was expressed in the recombinant yeast Pichia pastoris by using AOX1 promoter, in the presence of methanol, as carbon source and as a model for expression. This antibody is considered as a possible alternative for the diagnosis and treatment of atherosclerosis. An initial study was conducted in the production of the antibody fragment and the growth of yeast with two different work benches, varying the carbon source (glucose and glycerol). The results showed that the maximum cell concentrations, and the best production of antibody, were achieved with maintained working stock with glycerol as carbon source and preservation. Subsequently, a study was conducted in shaker and bioreactor, from a 23 complete factorial design, in which the variables studied were time induction (24, 28 and 32 hours), temperature induction (10, 20 and 30°C), and concentration of the inducer (methanol 0.5, 1.0 and 1.5%) on the response variable production of the antibody fragment. The only variable that had a statistically significant effect, with a confidence level of 95%, was the inducer concentration for the two production systems. The best yields of the antibody fragment were of 27.8 mg/L for the shaker and 41.3 mg/L for the bioreactor. The third study was to assess the production of antibody fragment into the shaker with the yeast at different carbon sources (sucrose, glycerol, glucose and sucrose/glucose 50%). In this study, the ANOVA analysis did not show statistically significant differences. We conducted planning to improve the quantification of antibody fragment in order to decrease the number of steps to avoid losses in the process. After getting a new protocol for the quantification, we carried out a new study to evaluate the production with different carbon sources. The analysis of the results showed that all carbon sources showed statistically significant effects, among which sucrose obtained the highest yield 93.7 mg/L of antibody fragment. These results were compared and analyzed through a microscope, in which with the observations of the yeast showed that the best antibody production was obtained when the yeast had sizes smaller than 0.895 microns. Next, a study was conducted to determine alternative sources of carbon and nitrogen (rice bran and wheat bran) to produce the antibody fragment in place of casein peptone and casaminoacids as substrates. The highest yield obtained in this case was 27.9 mg/L, when casamino was replaced by the wheat bran. Finally, we evaluated the effect of kLa in yeast growth and production of the antibody fragment scFv, the results showed that the best condition for the growth of the yeast was 96.12 h-1, on the other hand, the best condition for the production antibody fragment was 12.24 h-1 with a final relation yield of 8.12 mg/L antibody/g/L cell.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2007. / Adequate provision of oxygen to aerobic bioprocesses is essential for the optimisation of process kinetics. In bioprocesses in which the feedstock is an alkane, the supply of sufficient oxygen is of particular concern because the alkane molecular structure is deficient in oxygen. As a result, the oxygen demand has to be met solely by transfer of oxygen to the culture, necessitating a proportionately higher requirement for oxygen transfer. Maximisation of the rate of oxygen transfer is therefore of key importance in optimising the potential for alkane bioconversion, with respect to both operation and scale up. Nevertheless, the oxygen transfer rate (OTR), and its dependence on the overall volumetric mass transfer coefficient (KLa) in alkane-aqueous dispersions is not yet well understood. In view of the importance of an adequate OTR in the optimisation of alkane bioconversion, this study has focused on the identification and elucidation of the factors which underpin the behaviour of KLa in an alkane-aqueous dispersion. KLa behaviour was quantified in terms of the pressures imposed by turbulence and alkane fluid properties, through their influence on the Sauter mean diameter (D32), gas hold up, gas-liquid interface rigidity and gas-liquid interfacial area per unit volume. These properties were correlated with KLa over a wide range of agitation rates and alkane concentrations in alkane-aqueous dispersions. Experiments were conducted in a 5 litre aerated and agitated bioreactor at agitation rates of 600, 800, 1000 and 1200 rpm and alkane (n-C10-C13 cut) concentrations of 0, 2.5, 5, 10, and 20% (v/v). KLa determination was executed using both the gassing out and pressure step methods. The accuracy and reliability of these methods were compared under the full range of agitation rates and alkane concentrations. The pressure step method was conclusively shown to be superior provided that probe response was taken into account, and was therefore used in the correlations. The interfacial areas corresponding to the KLa values were calculated from the combined effects of D32 and gas hold up. D32 was determined from the measurement of the dispersed air bubble diameters by means of a photographic technique and image analysis. Image analysis was performed by a program that was developed in Matlab® using image acquisition and image processing techniques. This program used these techniques to extract information of the gas bubbles in the image. The gas hold up was determined using the dispersion height technique. The behaviour of KLa was shown to be dependent on both agitation and alkane concentration. Increasing agitation from 600 to 1200 rpm increased KLa for each of the alkane concentrations. The influence of agitation on the interfacial area was evaluated over the same range of agitation rates and the relationship between the corresponding KLa values and interfacial areas assessed. Increasing agitation rate similarly enhanced the interfacial area available for transfer for each of the alkane concentrations, resulting in the concomitant increase in KLa. This increase in interfacial area was related directly to a shear-induced decease in D32 and indirectly to an increased gas holdup as a result of the lower rise velocity of the smaller bubbles. In addition to the agitation, the presence of alkane markedly influenced KLa behaviour, but in different ways, depending on the alkane concentration. Alkane concentration between 2.5 and 5% (v/v) reduced D32 at constant agitation of 800, 1000 and 1200 rpm, a likely consequence of decreased surface tension and retarded coalescence conferred by the alkane. The smaller D32 and the consequential enhanced gas hold up served to amplify KLa through increased interfacial area. However, as alkane concentration was increased above 5% (v/v), the gas hold up decreased despite a continued decrease in D32, resulting in a corresponding decrease in both the interfacial area and KLa. This suggests that at the higher alkane concentrations, the influence of viscosity predominated, exerting multiple negative influences on the interfacial area and oxygen transfer coefficient. The trends were however, not observed at the low agitation of 600 rpm, where turbulence was significantly reduced and KLa was repressed for all alkane concentrations. The pressures imposed by turbulence and alkane properties on the interfacial area defined locales of KLa behaviour and three distinct KLa behavioural trends were identified, depending on the agitation rate and alkane concentration. Regime 1 was constrained between 2.5 and 5% (v/v) for agitation rates of 800 rpm and above. Here KLa enhancement was directly associated with increased interfacial area which was the major factor defining KLa in this regime. Regime 2 was constrained by alkane concentrations higher than 5% (v/v) for agitation rates of 800 rpm and above. In this regime, the KLa depression was observed with increasing alkane concentration suggesting a predominant influence of viscosity which would be likely to exert multiple negative influences on KLa, through both the interfacial area and KL. The interfacial area in this regime decreased mainly due to the negative effect of viscosity on gas holdup. Regime 3, characterised by a decline in KLa irrespective of the alkane concentration, occurred at agitation rates smaller than 800 rpm. It is likely that at low agitation rates, the contribution of turbulence was insufficient to exert a positive influence on the interfacial area In this regime, the interfacial decreased through the combined negative effect of increased D32 and decreased gas holdup. The resultant variation in OTR depended directly on the relative magnitudes of the KLa and oxygen solubility and indirectly on the process conditions which defined these magnitudes. Under conditions of enhanced KLa, OTR benefited from the combined increases in KLa and oxygen solubility. However, under conditions of KLa depression, the elevated oxygen solubility did not invariably outweigh the influence of KLa depression on OTR. Consequently, despite the considerably increased solubility of oxygen in alkane-based bioprocesses a potential decrease in OTR through depressed KLa underlines the critical importance of the quantification of this parameter in alkane-aqueous dispersions and the necessity for a definition of the locales of optimal KLa. Through the identification of the parameters which underpin the behaviour of KLa in alkane-aqueous dispersions and the quantification of the effect of process conditions on these parameters, a fundamental understanding of the KLa and OTR in alkane-aqueous dispersions has been developed. This provides a knowledge base for the prediction of optimal KLa in these systems and has wide application across all alkane-based bioprocesses.

A transferência de oxigênio é um fator limitante para grande parte dos cultivos em biorreatores que operam com organismos estritamente aeróbios devido à baixa solubilidade do oxigênio em meios de cultivo. A introdução de meios não convencionais, como polidimetilsiloxanos (PDMS), em biorreatores pode ser vista como uma melhoria para o ajuste desses processos. Este trabalho propõe uma investigação do emprego de polidimetilsiloxano fluido e emulsificado, usados como carreadores de oxigênio, em meios de cultivo na produção de lipases de Staphylococcus warneri EX17, cepa capaz de utilizar glicerol residual como substrato, visando melhorar a disponibilidade de oxigênio e aperfeiçoar a produtividade. Inicialmente, foram realizados estudos para selecionar o uso de glicerol residual, oriundo da síntese química de biodiesel como fonte de carbono. A atividade lipolítica foi semelhante com glicerol comercial e residual. Após a seleção do glicerol foram realizados experimentos em incubadora rotatória horizontal nas condições previamente otimizadas em trabalhos anteriores do grupo, adicionando PDMS. Dois tipos de PDMS foram testados, fluido e emulsificado. A produção de lipase foi significativamente maior no meio contendo PDMS. Assim, ferramentas de planejamento experimental, delineamento composto central (DCC) foram utilizadas para verificar a influencia do PDMS no coeficiente de transferência de oxigênio kLa em meio de cultivo produtor de lipase livre de células. O kLa aumentou significativamente no meio de cultivo contendo PDMS. Dois novos planejamentos foram desenhados, para otimizar a produção de lipase em meio de cultivo com células. Um para PDMS fluido e outro para PDMS emulsificado. No meio contendo PDMS fluido, a atividade lipolítica foi cinco vezes maior, enquanto que no meio contendo silicone emulsificado a atividade lipolítica foi três vezes maior. Este estudo demonstrou que a lipase de S. warneri EX17 pode ser produzida utilizando glicerol residual como fonte de carbono, e polidimetilsiloxanos como carreadores de oxigênio aumentando a transferência de oxigênio no meio e elevando a produção da enzima, que apresenta diversas possibilidades de aplicação, principalmente na indústria de alimentos. / The oxygen transfer is a limiting factor in many bioreactors cultivations, in which strictly aerobic organisms are grown due to the low solubility of oxygen in culture media. The introduction of unconventional media, such as polydimethylsiloxanes (PDMS) in bioreactors can be seen as a potential improvement for these processes. This work proposes an investigation of the use of polydimethylsiloxane in its two forms, fluid and emulsified, used as carriers of oxygen in culture media for the lipase production of Staphylococcus warneri EX17, strain capable of using glycerol as substrate, to improve the oxygen availability and improve enzyme productivity. Initially, studies were performed to select the use of residual glycerol, derived from the chemical synthesis of biodiesel as a source of carbon. The lipase activity was similar in both commercial and residual glycerol. Experiments were performed on orbital shaker, using conditions previously optimized in another work of the group, but with the addition of PDMS. Two types of PDMS were tested, the fluid and the emulsified. The lipase production was significantly higher in medium containing PDMS. Experimental design and central composite design (CCD) were used to evaluate the influence of PDMS on the oxygen transfer coefficient kLa in culture medium free of lipase-producing cells. The kLa increased significantly in the medium containing PDMS. Two new experimental plannings were designed to optimize the production of lipase in culture medium with cells. In the medium containing the PDMS fluid lipase activity was five times higher than medium without the oxygen carrier, while in the medium containing the emulsified silicone lipase activity was three times higher. This study showed that the lipase from S. warneri EX17 can be produced using residual glycerol as carbon source and that polydimethylsiloxanes works as interesting carrier of oxygen by increasing oxygen transfer rates, improving enzyme production, which has several possible applications, especially in the food industry.

A transferência de oxigênio é um fator limitante para grande parte dos cultivos em biorreatores que operam com organismos estritamente aeróbios devido à baixa solubilidade do oxigênio em meios de cultivo. A introdução de meios não convencionais, como polidimetilsiloxanos (PDMS), em biorreatores pode ser vista como uma melhoria para o ajuste desses processos. Este trabalho propõe uma investigação do emprego de polidimetilsiloxano fluido e emulsificado, usados como carreadores de oxigênio, em meios de cultivo na produção de lipases de Staphylococcus warneri EX17, cepa capaz de utilizar glicerol residual como substrato, visando melhorar a disponibilidade de oxigênio e aperfeiçoar a produtividade. Inicialmente, foram realizados estudos para selecionar o uso de glicerol residual, oriundo da síntese química de biodiesel como fonte de carbono. A atividade lipolítica foi semelhante com glicerol comercial e residual. Após a seleção do glicerol foram realizados experimentos em incubadora rotatória horizontal nas condições previamente otimizadas em trabalhos anteriores do grupo, adicionando PDMS. Dois tipos de PDMS foram testados, fluido e emulsificado. A produção de lipase foi significativamente maior no meio contendo PDMS. Assim, ferramentas de planejamento experimental, delineamento composto central (DCC) foram utilizadas para verificar a influencia do PDMS no coeficiente de transferência de oxigênio kLa em meio de cultivo produtor de lipase livre de células. O kLa aumentou significativamente no meio de cultivo contendo PDMS. Dois novos planejamentos foram desenhados, para otimizar a produção de lipase em meio de cultivo com células. Um para PDMS fluido e outro para PDMS emulsificado. No meio contendo PDMS fluido, a atividade lipolítica foi cinco vezes maior, enquanto que no meio contendo silicone emulsificado a atividade lipolítica foi três vezes maior. Este estudo demonstrou que a lipase de S. warneri EX17 pode ser produzida utilizando glicerol residual como fonte de carbono, e polidimetilsiloxanos como carreadores de oxigênio aumentando a transferência de oxigênio no meio e elevando a produção da enzima, que apresenta diversas possibilidades de aplicação, principalmente na indústria de alimentos. / The oxygen transfer is a limiting factor in many bioreactors cultivations, in which strictly aerobic organisms are grown due to the low solubility of oxygen in culture media. The introduction of unconventional media, such as polydimethylsiloxanes (PDMS) in bioreactors can be seen as a potential improvement for these processes. This work proposes an investigation of the use of polydimethylsiloxane in its two forms, fluid and emulsified, used as carriers of oxygen in culture media for the lipase production of Staphylococcus warneri EX17, strain capable of using glycerol as substrate, to improve the oxygen availability and improve enzyme productivity. Initially, studies were performed to select the use of residual glycerol, derived from the chemical synthesis of biodiesel as a source of carbon. The lipase activity was similar in both commercial and residual glycerol. Experiments were performed on orbital shaker, using conditions previously optimized in another work of the group, but with the addition of PDMS. Two types of PDMS were tested, the fluid and the emulsified. The lipase production was significantly higher in medium containing PDMS. Experimental design and central composite design (CCD) were used to evaluate the influence of PDMS on the oxygen transfer coefficient kLa in culture medium free of lipase-producing cells. The kLa increased significantly in the medium containing PDMS. Two new experimental plannings were designed to optimize the production of lipase in culture medium with cells. In the medium containing the PDMS fluid lipase activity was five times higher than medium without the oxygen carrier, while in the medium containing the emulsified silicone lipase activity was three times higher. This study showed that the lipase from S. warneri EX17 can be produced using residual glycerol as carbon source and that polydimethylsiloxanes works as interesting carrier of oxygen by increasing oxygen transfer rates, improving enzyme production, which has several possible applications, especially in the food industry.