O presente trabalho teve como objetivo avaliar a influência da intensidade de agitação e tipo de impelidor, bem como a estratégia de alimentação, em reator anaeróbio operado em batelada seqüencial, escala piloto (\'da ordem de\' 1 \'M POT.3\'), com agitação mecânica e em duas configurações: uma com biomassa granulada (ASBR) e outra com biomassa imobilizada em suporte inerte de espuma de poliuretano (ASBBR), aplicadas ao tratamento de esgoto doméstico. No estudo da intensidade de agitação e tipo de impelidor, para cada reator foram avaliadas três tipos de impelidores (turbina de pás planas, turbina de pás inclinadas 45º e hélice) associados a duas intensidades de agitação (40 e 80 rpm), obtendo uma combinação de 6 (seis) condições experimentais. A combinação da intensidade de agitação e tipo de impelidor que apresentou melhor desempenho no processo foi utilizado no estudo da estratégia de alimentação, na qual foi avaliada as seguintes condições: batelada alimentada durante 25%, 50%, 75% e 100% do ciclo. Os resultados obtidos permitiram concluir que: no ASBBR o aumento da intensidade de agitação de 40 rpm para 80 rpm permitiu uma melhoria nos fluxos de transferência de massa e, portanto, aumentou a velocidade de consumo de substrato; no ASBR o aumento da intensidade de agitação de 40 rpm para 80 rpm proporcinou uma desestabilização do sistema, provavelmente por causa da ruptura dos grânulos provocada pela maior agitação; os sistemas operados com impelidor do tipo hélice apresentaram vantagens, tais como: melhor eficiência de remoção de sólidos, maior valor da constante cinética de primeira ordem (melhor fluxo de transferência de massa e conseqüentemente maior consumo de substrato) e maior produção de alcalinidade, ou seja, maior estabilidade para o sistema; tanto o sistema ASBR como o ASBBR quando operados nas condições de batelada típica, batelada alimentada durante 50% e 75% do ciclo apresentaram melhores desempenhos no processo de tratamento, mostrando a flexibilidade operacional dos reatores anaeróbios operados em bateladas seqüenciais; e comparando os sistemas ASBBR e ASBR verificou-se que estes apresentaram comportamentos similares em todas as condições de operação de batelada alimentada avaliadas, não sendo possível, estatisticamente apontar um sistema com melhor desempenho. / The objective of this work was to assess the effect of the stirring speed, the type of impeller and the feed strategy in a mechanically stirred pilot-scale (\'da ordem de\' 1 \'M POT.3\') anaerobic sequencing batch reactor to two configurations: a containing granulated biomass (ASBR) and the other containing immobilized biomass (AnSBBR). Domestic wastewater was treated in 8-h cycles. Three impeller types (turbine with six-flat blades, turbine with six 45º-inclined blades and helix with three blades) were assessed at two different stirring speeds (40 and 80 rpm), totaling six experimental conditions. The stirring speed and the impeller that resulted in the best combination was used in work of the feed strategy . The reactors were operated at room temperature at four different feed strategies (fed batch during 25%, 50% and 75% of the cycle, and conventional fed-batch). The results allowed conclude that: in the AnSBBR increasing the stirring speed from 40 rpm to 80 rpm showed to improve mass transfer, with consequent increase in substrate consumption; in the ASBR increasing the stirring speed from 40 rpm to 80 rpm showed desestabilization in system, because of the disruption caused in the granules witth greater agitation; operation with the helix impeller showed some advantages over the turbine impellers, such as: improved efficiency in solids removal, higher value of the first order kinetic constant and higher alkalinity production; both for the ASBR as for the ASBBR the best performance in wastewater treatment was obtained when the reactors were operated at conventional batch, fed-batch during 50% and 75% of the cycle; no significant difference in performance was observed among these three conditions. Despite poor performance of the conventional fed-batch and fed-batch during 25% of the cycle compared to the other conditions, both these conditions presented operational stability. Hence, the anaerobic sequencing batch reactors presented operational flexibility as far as feed strategy is concerned.

O presente trabalho teve como objetivo avaliar a influência da intensidade de agitação e tipo de impelidor, bem como a estratégia de alimentação, em reator anaeróbio operado em batelada seqüencial, escala piloto (\'da ordem de\' 1 \'M POT.3\'), com agitação mecânica e em duas configurações: uma com biomassa granulada (ASBR) e outra com biomassa imobilizada em suporte inerte de espuma de poliuretano (ASBBR), aplicadas ao tratamento de esgoto doméstico. No estudo da intensidade de agitação e tipo de impelidor, para cada reator foram avaliadas três tipos de impelidores (turbina de pás planas, turbina de pás inclinadas 45º e hélice) associados a duas intensidades de agitação (40 e 80 rpm), obtendo uma combinação de 6 (seis) condições experimentais. A combinação da intensidade de agitação e tipo de impelidor que apresentou melhor desempenho no processo foi utilizado no estudo da estratégia de alimentação, na qual foi avaliada as seguintes condições: batelada alimentada durante 25%, 50%, 75% e 100% do ciclo. Os resultados obtidos permitiram concluir que: no ASBBR o aumento da intensidade de agitação de 40 rpm para 80 rpm permitiu uma melhoria nos fluxos de transferência de massa e, portanto, aumentou a velocidade de consumo de substrato; no ASBR o aumento da intensidade de agitação de 40 rpm para 80 rpm proporcinou uma desestabilização do sistema, provavelmente por causa da ruptura dos grânulos provocada pela maior agitação; os sistemas operados com impelidor do tipo hélice apresentaram vantagens, tais como: melhor eficiência de remoção de sólidos, maior valor da constante cinética de primeira ordem (melhor fluxo de transferência de massa e conseqüentemente maior consumo de substrato) e maior produção de alcalinidade, ou seja, maior estabilidade para o sistema; tanto o sistema ASBR como o ASBBR quando operados nas condições de batelada típica, batelada alimentada durante 50% e 75% do ciclo apresentaram melhores desempenhos no processo de tratamento, mostrando a flexibilidade operacional dos reatores anaeróbios operados em bateladas seqüenciais; e comparando os sistemas ASBBR e ASBR verificou-se que estes apresentaram comportamentos similares em todas as condições de operação de batelada alimentada avaliadas, não sendo possível, estatisticamente apontar um sistema com melhor desempenho. / The objective of this work was to assess the effect of the stirring speed, the type of impeller and the feed strategy in a mechanically stirred pilot-scale (\'da ordem de\' 1 \'M POT.3\') anaerobic sequencing batch reactor to two configurations: a containing granulated biomass (ASBR) and the other containing immobilized biomass (AnSBBR). Domestic wastewater was treated in 8-h cycles. Three impeller types (turbine with six-flat blades, turbine with six 45º-inclined blades and helix with three blades) were assessed at two different stirring speeds (40 and 80 rpm), totaling six experimental conditions. The stirring speed and the impeller that resulted in the best combination was used in work of the feed strategy . The reactors were operated at room temperature at four different feed strategies (fed batch during 25%, 50% and 75% of the cycle, and conventional fed-batch). The results allowed conclude that: in the AnSBBR increasing the stirring speed from 40 rpm to 80 rpm showed to improve mass transfer, with consequent increase in substrate consumption; in the ASBR increasing the stirring speed from 40 rpm to 80 rpm showed desestabilization in system, because of the disruption caused in the granules witth greater agitation; operation with the helix impeller showed some advantages over the turbine impellers, such as: improved efficiency in solids removal, higher value of the first order kinetic constant and higher alkalinity production; both for the ASBR as for the ASBBR the best performance in wastewater treatment was obtained when the reactors were operated at conventional batch, fed-batch during 50% and 75% of the cycle; no significant difference in performance was observed among these three conditions. Despite poor performance of the conventional fed-batch and fed-batch during 25% of the cycle compared to the other conditions, both these conditions presented operational stability. Hence, the anaerobic sequencing batch reactors presented operational flexibility as far as feed strategy is concerned.

Um reator anaeróbio tratando esgoto sintético de baixa concentração de matéria orgânica (500 mgDQO/L-1) e enriquecido com diferentes concentrações de sulfato (razões DQO/[SO4-2] de 1,34, 0,67 e 0,34) foi submetido à diferentes estratégias de alimentação (batelada e batelada alimentada de 3 h e de 6 h). O reator operou com capacidade de 4,0 L e tratou por ciclo de 8 h um volume de 2,0 L de esgoto sintético. Desta forma, executaram-se 9 condições diferentes que foram submetidas à análise com o objetivo de investigar a influência do tempo de alimentação e da razão DQO/[SO4-2] no desempenho do sistema. A temperatura do reator foi constante em 30 ± 1°C e a agitação mecânica foi fixada em 400 rpm. Como suporte para a imobilização da biomassa foram utilizados cubos de espuma de poliuretano. Os resultados obtidos demonstraram que a operação em batelada alimentada de 3 h com uma razão DQO/[SO4-2] de 0,34 apresentou as melhores condições para a remoção de matéria orgânica (89%). Quanto à remoção de sulfato observou-se uma estagnação neste mesmo modo de operação ao aumentar a concentração de sulfato no afluente, enquanto que na operação com alimentação em batelada alimentada de 6 h, a carga de sulfato removida (CSR) cresceu linearmente. Assim, a maior carga de sulfato removida foi registrada durante o ensaio em batelada alimentada de 6 h com uma razão DQO/[SO4-2] de 0,34, no qual foram removidos 0,55 g SO4-2(L.d). Com uma razão DQO/[SO4-2] de 1,34 e um tempo de enchimento de 6 h, o reator apresentou o melhor desempenho em termos de eficiência de remoção de sulfato (71%). Em todos os ensaios o reator apresentou estabilidade, com uma produção alta de alcalinidade a bicarbonato e a concentração de ácidos voláteis se manteve em níveis adequados. / An anaerobic sequencing batch reactor treating synthetic low strength wastewater (500 mgCOD/L-1) enriched with different sulfate concentrations (COD/[SO42] ratios of 1,34, 0,67 and 0,34) was operated at different fill times (batch mode and sequencing batch mode of 3 h and 6 h fill time). The reactor operated with a capacity of 4,0 L and treated per 8-h cycle 2,0 L of synthetic wastewater. Thus, 9 different configurations resulted from this configuration and were submitted under analyses with the objective to investigate the influence of each of those parameters on the performance of the system. The reactor was operated at a constant temperature of 30 ± 1°C and an mechanical agitation rate of 400 rpm. Cubic particles of polyurethane were used as support material for anaerobic biomass immobilization. The results showed that operating the reactor with a fill time of 3 h and a COD/[4-2] ratio of 0,34 was the most eficient strategy for COD removal (89%). During the same operation mode (3 h fill time) sulfate removal seemed to suffer stagnation due to the increasing sulfite concentation in the reactor, whereas with a fill time of 6 h the sulfate load removal increased linerally with increasing sulfate load. So the biggest removal, in terms of volumetric sulfate load, was obtained operating the reactor in sequencing batch mode with a fill time of 6 h and a COD/[4-2] ratio of 0,34. During this test the reactor removed 0,55 gSO4-2/(L.d) of 2,29 gSO4-2/(L.d) applied on the system. Operating at a COD/[SO4-2] ratio of 1,34 and a fill time of 6 h the reactor obtained the best results in terms of sulfate removal efficiency, with 71% of the sulfate removed from the system. In the entire period of analisis the reactor showed stability with a suficient production of alkalinity to maintain the concentration of volatil acids in adequate levels.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work aimed to study the enzymatic hydrolysis of malt bagasse, using mechanical agitation, ultrasonic probe and supercritical CO2. Enzymatic hydrolysis was performed on granular starch. For this, was used a commercial amylolytic complex suitable for this type of hydrolysis, STARGENTM 002. For each technology were studied different variables. The first planning was carried out for the hydrolysis assisted by mechanical agitation and the variables studied were temperature (°C), enzyme concentration (%, m/m) and substrate concentration in the medium (m/m). TRS concentrations were found up to 75.5 g per kg of substrate and all variables had a significant effect on the response. This concentration of TRS was defined as mass yield of the process and this yield was corroborated by a kinetic, developed under the same conditions, with slight increase in temperature (70°C). From this initial planning, the temperature variables (70°C), enzyme concentration (8.2%) and substrate concentration (170 g.L-1) were fixed to carry out the hydrolysis assisted by direct and indirect ultrasound. The variables were investigated in the second planning were amplitude (%) and pulse factor (-) for 2 hours of reaction. With the application of direct sonication, it was possible to achieve 100% efficiency in the starch conversion process. The TRS for the best essay (5) was 370.86 g.kg-1. For indirect sonication TRS concentration at the best condition (run 6) was 162.96 g / kg of substrate. A kinetic assay for the best condition under direct sonication was carried out for 3 hours, confirming that the ultrasound increases the reaction rate resulting in better yields in less time compared to the other techniques. For reactions with supercritical CO2 was studied the influence of the moisture content, temperature and pressure, where the best result among all the reactions was using at pressure 175 bar, 40 °C for temperature and moisture content of 80%, resulting in 104.28 g of TRS per kg of dry pulp. Ultrasound showed better results than other technologies investigated in this study. / Este trabalho teve como objetivo estudar a hidrólise enzimática do bagaço de malte, utilizando agitação mecânica, sonda de ultrassom e CO2 supercrítico. A hidrólise enzimática foi realizada sobre o amido granular. Para tanto, foi utilizado um complexo amilolítico comercial próprio para este tipo de hidrólise, STARGENTM 002. Para cada tecnologia foram estudadas diferentes variáveis. O primeiro planejamento foi realizado para a hidrólise assistida por agitação mecânica e as variáveis estudadas foram temperatura (ºC), concentração de enzima (%, m/m) e concentração de substrato no meio (m/m). Foram encontradas concentrações de ART de até 75,5 g por kg de substrato e todas as variáveis apresentaram efeito significativo sobre a resposta. Essa concentração de ART, foi definida como rendimento mássico do processo e este valor foi corroborado com uma cinética, desenvolvida nas mesmas condições do melhor ensaio, com leve aumento apenas na temperatura (70ºC), e indicou valor concernente ao anterior além de provar que o tempo de 4 horas de hidrólise foi suficiente. A partir deste primeiro planejamento, as variáveis temperatura (70°C), concentração de enzima (8,2%) e concentração de substrato (170 g.L-1) foram fixadas para a realização da hidrólise assistida por ultrassom, de forma direta e indireta. As variáveis investigadas neste segundo planejamento foram amplitude (%) e fator de pulso (-) durante 2 horas de reação. Com aplicação de sonicação direta, foi possível alcançar 100% de eficiência no processo de conversão do amido. A resposta encontrada para o melhor ensaio (5) foi de 370,86 g.kg-1. Já com aplicação de sonicação indireta, a eficiência do processo caiu pela metade e o melhor resultado foi para o ensaio de número 6, com concentração de 162,96 g ART/ kg de substrato. Uma cinética para o melhor ensaio de sonicação direta foi desenvolvida durante 3 horas, atestando que o ultrassom aumenta a velocidade da reação resultando no melhor rendimento em menor tempo comparado às outras técnicas. Para as reações com CO2 supercrítico, foi estudada a influência da umidade, da temperatura e pressão, onde o melhor resultado obtido, entre todas as reações, foi com a utilização de pressão de 175 bar, temperatura de 40ºC e 80% de água adicionada ao bagaço, resultando em 104,28 g de ART por kg de bagaço seco. Esse valor corresponde a 11,53% de eficiência da reação de hidrólise do amido em açúcares redutores totais. De um modo geral, os processos obtiveram um bom desempenho na obtenção de açúcares fermentescíveis, destacando-se o ultrassom em relação as demais tecnologias testadas.

Um reator anaeróbio tratando esgoto sintético de baixa concentração de matéria orgânica (500 mgDQO/L-1) e enriquecido com diferentes concentrações de sulfato (razões DQO/[SO4-2] de 1,34, 0,67 e 0,34) foi submetido à diferentes estratégias de alimentação (batelada e batelada alimentada de 3 h e de 6 h). O reator operou com capacidade de 4,0 L e tratou por ciclo de 8 h um volume de 2,0 L de esgoto sintético. Desta forma, executaram-se 9 condições diferentes que foram submetidas à análise com o objetivo de investigar a influência do tempo de alimentação e da razão DQO/[SO4-2] no desempenho do sistema. A temperatura do reator foi constante em 30 ± 1°C e a agitação mecânica foi fixada em 400 rpm. Como suporte para a imobilização da biomassa foram utilizados cubos de espuma de poliuretano. Os resultados obtidos demonstraram que a operação em batelada alimentada de 3 h com uma razão DQO/[SO4-2] de 0,34 apresentou as melhores condições para a remoção de matéria orgânica (89%). Quanto à remoção de sulfato observou-se uma estagnação neste mesmo modo de operação ao aumentar a concentração de sulfato no afluente, enquanto que na operação com alimentação em batelada alimentada de 6 h, a carga de sulfato removida (CSR) cresceu linearmente. Assim, a maior carga de sulfato removida foi registrada durante o ensaio em batelada alimentada de 6 h com uma razão DQO/[SO4-2] de 0,34, no qual foram removidos 0,55 g SO4-2(L.d). Com uma razão DQO/[SO4-2] de 1,34 e um tempo de enchimento de 6 h, o reator apresentou o melhor desempenho em termos de eficiência de remoção de sulfato (71%). Em todos os ensaios o reator apresentou estabilidade, com uma produção alta de alcalinidade a bicarbonato e a concentração de ácidos voláteis se manteve em níveis adequados. / An anaerobic sequencing batch reactor treating synthetic low strength wastewater (500 mgCOD/L-1) enriched with different sulfate concentrations (COD/[SO42] ratios of 1,34, 0,67 and 0,34) was operated at different fill times (batch mode and sequencing batch mode of 3 h and 6 h fill time). The reactor operated with a capacity of 4,0 L and treated per 8-h cycle 2,0 L of synthetic wastewater. Thus, 9 different configurations resulted from this configuration and were submitted under analyses with the objective to investigate the influence of each of those parameters on the performance of the system. The reactor was operated at a constant temperature of 30 ± 1°C and an mechanical agitation rate of 400 rpm. Cubic particles of polyurethane were used as support material for anaerobic biomass immobilization. The results showed that operating the reactor with a fill time of 3 h and a COD/[4-2] ratio of 0,34 was the most eficient strategy for COD removal (89%). During the same operation mode (3 h fill time) sulfate removal seemed to suffer stagnation due to the increasing sulfite concentation in the reactor, whereas with a fill time of 6 h the sulfate load removal increased linerally with increasing sulfate load. So the biggest removal, in terms of volumetric sulfate load, was obtained operating the reactor in sequencing batch mode with a fill time of 6 h and a COD/[4-2] ratio of 0,34. During this test the reactor removed 0,55 gSO4-2/(L.d) of 2,29 gSO4-2/(L.d) applied on the system. Operating at a COD/[SO4-2] ratio of 1,34 and a fill time of 6 h the reactor obtained the best results in terms of sulfate removal efficiency, with 71% of the sulfate removed from the system. In the entire period of analisis the reactor showed stability with a suficient production of alkalinity to maintain the concentration of volatil acids in adequate levels.

Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.