O poli(3-hidroxibutirato) é um biopolímero obtido a partir de fontes renováveis e é um poliéster biodegradável. Possui um grande potencial de aplicação principalmente em artefatos e embalagens descartáveis e também na área médica, devido sua biocompatibilidade. Neste trabalho foi estudada a produção de P(3HB) por Bacillus megaterium em meio mineral, buscando o aumento na produção deste biopolímero, bem como representar matematicamente este bioprocesso. Foram avaliadas a influência do pH e da disponibilidade de oxigênio em cultivos utilizando sacarose como fonte de carbono. Também foi avaliada a influência de diferentes fontes de nitrogênio, com glicerol ou sacarose como fonte de carbono. A influência do pH foi estudada mediante experimentos em biorreator de 4 L com controle da concentração de oxigênio dissolvido acima de 40 % em relação a saturação, com ou sem controle de pH. Observou-se que o acúmulo de P(3HB) foi maior quando o pH não foi controlado, 34 % em relação a biomassa em comparação com 28 % quando pH foi controlado. Foi realizada a modelagem da produção de P(3HB), com proposição de quatro modelos. Os dados experimentais dos cultivos com e sem controle de pH foram utilizados na etapa de estimação de parâmetros. Em comparação com modelos relatados na literatura os modelos propostos apresentaram melhor ajuste, com menor ou igual número de parâmetros, e forneceram estimativas dos parâmetros com menor variabilidade. A avaliação da influência do oxigênio foi realizada através de cultivos em biorreator de 4 L, sem controle de pH, com diferentes condições de transferência de oxigênio, obtidas variando-se a rotação do impelidor entre 100 rpm e 600 rpm. O maior acúmulo de biopolímero foi obtido em experimento com rotação do impelidor 200 rpm, o qual apresentou um baixo coeficiente volumétrico de transferência de oxigênio (kLa = 0,006 s-1) e possibilitou acúmulo de 62 % de P(3HB) em relação a biomassa seca. Para avaliar a melhor fonte de nitrogênio, foram realizados experimentos em estufa incubadora rotatória com cinco fontes de nitrogênio diferentes: cloreto de amônio, citrato de amônio, sulfato de amônio, fosfato de amônio e nitrato de amônio. Com relação à fonte de carbono, sacarose possibilitou maior acúmulo de P(3HB) e biomassa que glicerol. Com relação à fonte de nitrogênio, cloreto de amônio possibilitou maior acúmulo de P(3HB) e biomassa quando utilizado glicerol como fonte de carbono, enquanto que sulfato de amônio aumenta a produção com sacarose. / Poly(3-hydroxybutyrate) is a biodegradable polyester produced from renewable resources. This polymer has a great potential to be used as disposable items and packaging, and also in the medical field due to its biocompatibility. In this work, the P(3HB) production by Bacillus megaterium in mineral medium was studied aiming the increase of biopolymer production, as well the development of a mathematical model for this bioprocess. The influence of pH and oxygen transfer rate in a B. megaterium culture using sucrose as carbon source was evaluated, and the influence of different nitrogen sources using sucrose or glycerol as carbon source was analyzed. The influence of pH-medium was studied in bench-scale bioreactor under controlled and uncontrolled pH. The dissolved oxygen was controlled above 40 % of saturation. It was observed that P(3HB) accumulation was higher in the experiment without pH control (34 % of dry biomass) than in the experiment with pH control (28 % of dry biomass). Four models were proposed to perform the mathematical modeling of this bioprocess. The experimental data of the cultures with and without pH control were used for parameter estimation. Compared to earlier models reported in the literature, the proposed models show better fitting, with less or equal number of parameters, and provide parameters estimative with much lower variability. The evaluation of oxygen transfer rate influence was performed in 4 liters bioreactor cultures without pH control, with different conditions of oxygen transfer rates by ranging impeller speed between 100 rpm and 600 rpm. The higher P(3HB) accumulation (62 % of dry biomass) was attained in the experiment with low volumetric oxygen transfer rate (kLa = 0.006 s-1), at 200 rpm impeller speed. To evaluate the best nitrogen source for sucrose or glycerol as carbon sources, experiments were performed in shaker with five different nitrogen sources: ammonium chloride, ammonium citrate, ammonium sulfate, ammonium phosphate and ammonium nitrate. With respect to carbon source, sucrose allowed higher biomass and P(3HB) accumulation than glycerol. With respect to nitrogen source, ammonium chloride allowed highest biomass and P(3HB) accumulation when glycerol was used as carbon source, whereas ammonium sulfate showed the best results for sucrose.

No description available.

Presented work is focused on study of polyhydroxybutyrate production in bacteria. In theoretical part short characterization of PHB was given and the most common representative of wide group of polyhydroxyalkanoates (PHA) were described. Then, production of PHB and copolymer P(HB-co-HV) in selected bacterial strains was experimentally proven. First, PHB production in Bacillus megaterium using synthetic medium was studied. The PHB content in cells was increased during cultivation under limiting conditions, despite low growth. Addition of ethanol into production media resulted in increased PHB synthesis as well as biomass production (21 % PHB of 1,8 g/l biomass). Further, BM medium containing 8 g/l glucose was tested. PHB production was more than 1 g/l at significant growth increase when compared with synthetic medium. The bacteria B.megaterium showed, except glucose, ability to utilize maltose and xylose. Another cultivations were tested with bacterial strain Azotobacter vinelandii, which is capable of copolymer P(HB-co-HV) synthesis. Maximal growth and copolymer content was reached on Burk's medium with 30 g/l of glucose. Addition of peroxide to growth medium influenced P(HB-co-HV) synthesis to 46 % of 2,6 g/l biomass. Bacteria A.vinelandii showed the best growth on maltose, even compared with glucose (54 % copolymer of biomass content). Finally, PHB production on industrial waste product – whey was monitored. Using Plackett-Burman design for statistical media optimization, the whey content was modified. B.megaterium grown on adjusted whey reached 0,5 g/l PHB, 32 % of cell's content.

No description available.

A recombinant Bacillus megaterium strain with a chromosomally integrated gene kerA, expressing keratinase, was analyzed regarding action in specific environmental stresses to obtain optimum parameters necessary for bacterial growth and optimal expression of the recombinant gene.The strain was grown on chicken feathers as substrate and the degradation of feather keratin during the growth was investigated with different levels of pH, temperature and feather concentration in the broth. Two strains were analyzed, capable to grow with 575 x and 1025 x amounts of antibiotics, erythromycin, where the strain of 1025 x showed higher potential for expression. Maximum keratinolytic activity was achieved at a pH of value 8.5, at temperature of 45 °C and substrate concentration of 0.2 % (w/v). The growth rate was determined by total plate count, which indicated that there was an increasing number of colony forming units together with increasing concentration of soluble keratin. The diluted fractions did not appear to produce colonies to any significant amount. However the results for which reason the results for optimal growth conditions are guidelines for future analysis for a chromosomally recombinant Bacillus megaterium. / Program: Master of Science with a Major in Resource Recovery – Industrial Biotechnology

Biopolímeros polihidroxialcanoatos (PHAs) são poliésteres sintetizados por inúmeras bactérias e armazenados em corpos de inclusão nas células como reserva de energia, são produzidos a partir de fontes renováveis, e sua principal característica é a biodegradabilidade. Neste trabalho realizou-se a produção do PHA poli(3- hidroxibutirato) (P3HB) em cultivos submersos da bactéria Bacillus megaterium em meio mineral, utilizando-se sacarose como fonte de carbono e sulfato de amônio como fonte de nitrogênio. Esta bactéria apresenta comportamento diferente das bactérias comumente utilizadas para produção de biopolímero, sendo capaz de produzir grandes quantidades de P(3HB) sem limitação de nitrogênio. Experimentos em estufa incubadora rotatória foram realizados, segundo planejamento fatorial 22 composto central rotacional e três repetições no ponto central, a fim de se avaliar a melhor concentração inicial de sacarose e a razão inicial carbono/nitrogênio. Nestes experimentos, obteve-se um acúmulo de P(3HB) nas células de cerca de 75%. Não houve limitação de nitrogênio e sacarose para a maioria dos casos e observou-se uma forte correlação entre o acúmulo de P(3HB) e o pH do meio. Em biorreator foram realizados experimentos com controle de oxigênio dissolvido e pH, e com a condição de substrato inicial escolhida em função do planejamento de experimentos realizado. Obteve-se acúmulo de P(3HB) de 30%, inferior ao obtido em estufa incubadora rotatória. Modelos fenomenológicos de cinética microbiana, produção de polímero e consumo de substratos foram estudados, propondo-se modificações. Os parâmetros dos modelos foram estimados com os dados obtidos experimentalmente no biorreator. O modelo proposto obteve um desempenho superior aos demais estudados e com um número menor de parâmetros. Simulações foram realizadas a fim de verificar o comportamento do modelo frente a variações nas condições iniciais. / Polyhydroxyalkanoates (PHAs) biopolymers are polyesters synthesized by numerous bacteria and are accumulated on inclusion bodies in the cytoplasm of the cells as energy storage, they are produced from renewable carbon sources and its manly characteristic is the biodegradability. In this work it was carried the production of PHA poly(3-hydroxybutyrate) (P3HB) out in submerse cultures of bacteria Bacillus megaterium in the mineral medium, using sucrose as carbon source and ammonium sulphate as nitrogen source. This bacteria shows different behavior from bacteria commonly used on biopolymer production, and it is able to produce large amounts of P(3HB) without nitrogen limitation. Shaker experiments were realized, according to a central composite design and three repetitions in the central point, in order to evaluate the best initial sucrose concentration and carbon-to-nitrogen ratio. In these experiments, P(3HB) was accumulated in the cell about 75%. In most of the cases, there was no limitation in the sucrose or nitrogen, and a strong correlation between P(3HB) accumulated and the medium pH was observed. Bioreactor experiments were carried out with controlled dissolved oxygen and pH. Substrate initial concentration was chosen according to the experiment design. P(3HB) accumulation in bioreactor was lower than in shaker, about 30%. Phenomenological models of microbial kinetics, polymer production, and substrate consumption were studied and improvements were proposed. The models parameters were estimated using the experiment data from the bioreactor. The proposed model showed a better performance than the available models, with a smaller number of parameters. Simulations were performed with the purpose of verifying the model behavior for initial conditions changes.

Biopolímeros polihidroxialcanoatos (PHAs) são poliésteres sintetizados por inúmeras bactérias e armazenados em corpos de inclusão nas células como reserva de energia, são produzidos a partir de fontes renováveis, e sua principal característica é a biodegradabilidade. Neste trabalho realizou-se a produção do PHA poli(3- hidroxibutirato) (P3HB) em cultivos submersos da bactéria Bacillus megaterium em meio mineral, utilizando-se sacarose como fonte de carbono e sulfato de amônio como fonte de nitrogênio. Esta bactéria apresenta comportamento diferente das bactérias comumente utilizadas para produção de biopolímero, sendo capaz de produzir grandes quantidades de P(3HB) sem limitação de nitrogênio. Experimentos em estufa incubadora rotatória foram realizados, segundo planejamento fatorial 22 composto central rotacional e três repetições no ponto central, a fim de se avaliar a melhor concentração inicial de sacarose e a razão inicial carbono/nitrogênio. Nestes experimentos, obteve-se um acúmulo de P(3HB) nas células de cerca de 75%. Não houve limitação de nitrogênio e sacarose para a maioria dos casos e observou-se uma forte correlação entre o acúmulo de P(3HB) e o pH do meio. Em biorreator foram realizados experimentos com controle de oxigênio dissolvido e pH, e com a condição de substrato inicial escolhida em função do planejamento de experimentos realizado. Obteve-se acúmulo de P(3HB) de 30%, inferior ao obtido em estufa incubadora rotatória. Modelos fenomenológicos de cinética microbiana, produção de polímero e consumo de substratos foram estudados, propondo-se modificações. Os parâmetros dos modelos foram estimados com os dados obtidos experimentalmente no biorreator. O modelo proposto obteve um desempenho superior aos demais estudados e com um número menor de parâmetros. Simulações foram realizadas a fim de verificar o comportamento do modelo frente a variações nas condições iniciais. / Polyhydroxyalkanoates (PHAs) biopolymers are polyesters synthesized by numerous bacteria and are accumulated on inclusion bodies in the cytoplasm of the cells as energy storage, they are produced from renewable carbon sources and its manly characteristic is the biodegradability. In this work it was carried the production of PHA poly(3-hydroxybutyrate) (P3HB) out in submerse cultures of bacteria Bacillus megaterium in the mineral medium, using sucrose as carbon source and ammonium sulphate as nitrogen source. This bacteria shows different behavior from bacteria commonly used on biopolymer production, and it is able to produce large amounts of P(3HB) without nitrogen limitation. Shaker experiments were realized, according to a central composite design and three repetitions in the central point, in order to evaluate the best initial sucrose concentration and carbon-to-nitrogen ratio. In these experiments, P(3HB) was accumulated in the cell about 75%. In most of the cases, there was no limitation in the sucrose or nitrogen, and a strong correlation between P(3HB) accumulated and the medium pH was observed. Bioreactor experiments were carried out with controlled dissolved oxygen and pH. Substrate initial concentration was chosen according to the experiment design. P(3HB) accumulation in bioreactor was lower than in shaker, about 30%. Phenomenological models of microbial kinetics, polymer production, and substrate consumption were studied and improvements were proposed. The models parameters were estimated using the experiment data from the bioreactor. The proposed model showed a better performance than the available models, with a smaller number of parameters. Simulations were performed with the purpose of verifying the model behavior for initial conditions changes.

Biopolímeros polihidroxialcanoatos (PHAs) são poliésteres sintetizados por inúmeras bactérias e armazenados em corpos de inclusão nas células como reserva de energia, são produzidos a partir de fontes renováveis, e sua principal característica é a biodegradabilidade. Neste trabalho realizou-se a produção do PHA poli(3- hidroxibutirato) (P3HB) em cultivos submersos da bactéria Bacillus megaterium em meio mineral, utilizando-se sacarose como fonte de carbono e sulfato de amônio como fonte de nitrogênio. Esta bactéria apresenta comportamento diferente das bactérias comumente utilizadas para produção de biopolímero, sendo capaz de produzir grandes quantidades de P(3HB) sem limitação de nitrogênio. Experimentos em estufa incubadora rotatória foram realizados, segundo planejamento fatorial 22 composto central rotacional e três repetições no ponto central, a fim de se avaliar a melhor concentração inicial de sacarose e a razão inicial carbono/nitrogênio. Nestes experimentos, obteve-se um acúmulo de P(3HB) nas células de cerca de 75%. Não houve limitação de nitrogênio e sacarose para a maioria dos casos e observou-se uma forte correlação entre o acúmulo de P(3HB) e o pH do meio. Em biorreator foram realizados experimentos com controle de oxigênio dissolvido e pH, e com a condição de substrato inicial escolhida em função do planejamento de experimentos realizado. Obteve-se acúmulo de P(3HB) de 30%, inferior ao obtido em estufa incubadora rotatória. Modelos fenomenológicos de cinética microbiana, produção de polímero e consumo de substratos foram estudados, propondo-se modificações. Os parâmetros dos modelos foram estimados com os dados obtidos experimentalmente no biorreator. O modelo proposto obteve um desempenho superior aos demais estudados e com um número menor de parâmetros. Simulações foram realizadas a fim de verificar o comportamento do modelo frente a variações nas condições iniciais. / Polyhydroxyalkanoates (PHAs) biopolymers are polyesters synthesized by numerous bacteria and are accumulated on inclusion bodies in the cytoplasm of the cells as energy storage, they are produced from renewable carbon sources and its manly characteristic is the biodegradability. In this work it was carried the production of PHA poly(3-hydroxybutyrate) (P3HB) out in submerse cultures of bacteria Bacillus megaterium in the mineral medium, using sucrose as carbon source and ammonium sulphate as nitrogen source. This bacteria shows different behavior from bacteria commonly used on biopolymer production, and it is able to produce large amounts of P(3HB) without nitrogen limitation. Shaker experiments were realized, according to a central composite design and three repetitions in the central point, in order to evaluate the best initial sucrose concentration and carbon-to-nitrogen ratio. In these experiments, P(3HB) was accumulated in the cell about 75%. In most of the cases, there was no limitation in the sucrose or nitrogen, and a strong correlation between P(3HB) accumulated and the medium pH was observed. Bioreactor experiments were carried out with controlled dissolved oxygen and pH. Substrate initial concentration was chosen according to the experiment design. P(3HB) accumulation in bioreactor was lower than in shaker, about 30%. Phenomenological models of microbial kinetics, polymer production, and substrate consumption were studied and improvements were proposed. The models parameters were estimated using the experiment data from the bioreactor. The proposed model showed a better performance than the available models, with a smaller number of parameters. Simulations were performed with the purpose of verifying the model behavior for initial conditions changes.

Philosophiae Doctor - PhD / The popularization and health benefits associated with the "Mediterranean diet" saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds.

On July 10, 1968, while observing the effects of ultra-violet light on a plate culture of Sarcino lutea, o contaminant was found growing in the form of a colony surrounded by a clear zone of inhibition where S. lutea had failed to grow. A smear from this contaminant colony was found to contain a Gram positive, chain-ond spore-forming bacillus. The organism was isolated and its antagonism against S. lutea was reshocked. Preliminary investigation of its activity against other bacteria indicated that this activity was directed against 5. lutea only. I was decided to continue the investigations with two aims in mind.