The subject of this diploma thesis is the study of the production of polyhydroxyalkanoates (PHA) employing Cupriavidus malaysiensis. The aim of this thesis is to obtain and characterize the polymer with the most advantageous properties. The theoretical part deals with polyhydroxyalkanoates and their biotechnological production. Then selected microorganisms producing 3-hydroxybutyrate and 4 hydroxybutyrate, P(3HB co 4HB), are described. After that, the theoretical part deals with the production of PHA employing Cupriavidus malaysiensis. The bacterial strains of Cupriavidus malaysiensis DSM 19416, 19379 and 25816 were used in the experimental part. The strain Cupriavidus necator H16 was used as a control strain. First, the ability to utilize various carbon sources by the given bacterial strains was determined. Then, the aim was to select the optimal carbon source, a precursor for the synthesis of a polymer with high content of 4-hydroxybutyrate, and to determine the ideal culturing conditions for the growth of the microorganisms, which have been studied, and the production of P(3HB co-4HB). A two-stage culture and bioreactor culture was also performed to produce the desired copolymer. Furthermore, the production of terpolymer of 3-hydroxybutyrate, 4 hydroxybutyrate and 3-hydroxyvalerate, P(3HB co 4HB co 3HV), was studied by single stage and two-stage cultivation. In the last part, the produced polymers were characterized by gas chromatography with flame ionization detector (GC-FID), size exclusion chromatography with multi-angle static light scattering (SEC-MALS), differential scanning calorimetry (DSC) and thermogravimetry.

Made available in DSpace on 2016-12-08T17:19:29Z (GMT). No. of bitstreams: 1 Elementos pre-textuais.pdf: 89508 bytes, checksum: 6cbceb5e70da4a660d5066d97a20334f (MD5) Previous issue date: 2007-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the past two decades, polyhydroxyalkanoates (PHAs) have been the focus of extensive research considering their potential application as biocompatible and biodegradable thermoplastics, due to their hydrolyzability in the human body as well as in natural circumstances. Aiming the development of new ductile and biodegradable materials, systems of poly(3-hydroxybutyrate), P(3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HV-co-3HV) with poly(e-caprolactone) triol (PCL-T, 900 and 300 g.mol-1) with addition of 0 to 30 % of PCL-T (w/w), were obtained by casting or injection molding. The thermal behavior, the morphology and the crystallinity of these mixtures were studied by differential scanning calorimetry (DSC), thermogravimetric analysis (TG), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR). The rheology and the melt processing of P(3HB)/PCL-T systems were also studied. The crystallinity degree of casted P(3HB) decreased from 68 to 49% with addition of 5% of PCL-T 900, and from 68% to 54% with 5% of PCL-T 300 content. The TG analysis showed that the PCL-T did not change the thermal stability of P(3HB). It was also observed by DSC that the Tm of PCL-T did not change in all compositions, while the Tm of P(3HB) decreased when PCL-T content was increased. It was also noticed that the Tg of P(3HB) increased from 2.75 to -2ºC with the increase of PCL-T 900 content. The Tg of P(3HB) change from 2.75 to -10ºC with the PCL-T 300 content. Indeed, FTIR analyses did not show evidences of hydrogen bonds between the polymers, while SEM analysis revealed porous films. A similar behavior was observed for P(3HB-co-3HV)/PCL-T 900 systems, while no change in the cristallinity degree was observed for P(3HB-co-3HV)/PCL-T 300 systems. For blends with P(3HB-co-3HV), DSC analyses revealed that the Tg of P(3HB-co-3HV) decreased from 2ºC to ca. -10ºC with the addition of 15wt% of PCL-T 900 or PCL-T 300. It is also noticed that the Tm of P(3HB-co- 3HV) and PCL-T decreases gradually as the PCL-T content is increased. Rheological analyses showed a pseudoplastic behavior for all the samples and a very significant decrease in the viscosity when the PCL-T content was increased. The P(3HB-co-3HV)/PCL-T 300 systems with 20 and 30% of PCL-T 300 contents showed a behavior look likes a Newtonian. Tensile tests of injected specimens showed an increase of 67% in the elongation at break with 5% of PCL-T, while the Young modulus decreased 7.5%. A higher PCL-T content (10%), however, decreases the modulus more significantly (50% in relation to P(3HB)). The crystallinity degree of P(3HB), obtained from XRD analyses, decreases with the addition of PCL-T (from 59 to 50% with 10% of PCL-T) and is in agreement with tensile tests and SEM results, that presented a higher ductility for the mixtures with PCL-T. TG analyses showed that the onset temperature does not vary significantly with the PCL-T content. FTIR spectra present interesting changes with PCL-T addition that are possibly related to interactions between the polymers. The better processing behavior of the PHAs with the addition of PCL-T suggests that the latter acts both as a plasticizer and a flow auxiliary, providing new, more flexible biodegradable materials. / Nas últimas duas décadas, os polihidroxialcanoatos (PHAs) foram o foco de pesquisas extensivas considerando suas potenciais aplicações como termoplásticos biocompatíveis e biodegradáveis, devido a sua hidrolisidade no corpo humano bem como em circunstâncias naturais. Objetivando o desenvolvimento de novos materiais dúcteis e biodegradáveis, sistemas de poli(3-hidroxibutirato), P(3HB) e poli(3-hidroxibutirato-co-3-hidroxivalerato), P(3HB-co-3HV) com poli(å-caprolactona) triol, PCL-T (300 e 900g.mol-1) com adição de 0 a 30% de PCL-T (m/m), foram obtidos por casting ou moldagem por injeção. O comportamento térmico, a morfologia e a cristalinidade destas misturas foram estudados, para isso foram utilizadas as seguintes técnicas: calorimetria exploratória diferencial (DSC), análise termogravimétrica (TG), microscopia eletrônica de varredura (SEM), difração de raios-X (XRD) e espectroscopia no infravermelho por transformada de Fourier (FTIR). A reologia e o processamento dos sistemas de P(3HB) /PCL-T também foram estudados. O grau de cristalinidade do P(3HB) caiu de 68 para 49% com adição de 5% de PCL-T 900, e de 68 para 54% com adição de PCL-T 300. A análise de TG mostrou que a PCL-T não interfere na estabilidade térmica do P(3HB). Foi também observado por DSC que a Tm da PCL-T não mudou em todas as composições, enquanto que a Tm do P(3HB) decresceu quando o teor de PCL-T aumentou. Notou-se também que a Tg do P(3HB) diminuiu de 2,75 para -2ºC com o aumento da PCL-T 900. A Tg do P(3HB) mudou de 2,75 para -10ºC com o aumento do teor de PCL-T 300. De fato, as análises de FTIR não mostraram evidencias de ligações de hidrogênio entre os polímeros, enquanto as análises de SEM revelaram filmes porosos. Um comportamento similar foi observado para o sistema de P(3HB-co-3HV)/PCL-T 900, enquanto nenhuma mudança no grau de cristalinidade do sistema de P(3HB-co-3HV)/PCL-T 300 foi observada. Para blendas com P(3HB-co-3HV), as análises de DSC revelaram que a Tg do P(3HB-co-3HV) diminuiu de 2ºC para ca. -10ºC com adição de 15% de PCL-T 900 PCL-T 300. Notou-se também que a Tm do P(3HB-co-3HV) e da PCL-T diminuíram ou gradualmente com o aumento do teor de PCL-T. A análise reologica mostrou um comportamento pseudoplastico para todas as amostras e um decréscimo muito significativo na viscosidade quando a PCL-T e adicionada. Os sistemas de P(3HB-co-3HV)/PCL-T 300 nas composições 80/20 e 85/15 mostraram um comportamento muito próximo do Newtoniano. Os testes de resistência à tração das espécies injetadas mostraram um aumento de 67% no alongamento com 5% de PCL-T, enquanto que o modulo de elasticidade diminuiu cerca de 7,5%. Uma alta quantidade de PCL-T (10%), entretanto, diminuiu o modulo mais significativamente (50% em relação ao P(3HB)). O grau de cristalinidade do P(3HB), obtido por XRD, diminuiu com adição de PCL-T 900 (de 58 para 51% com 10% de PCL-T 900) concordando com os testes de resistência a tração e resultados de SEM, que apresentaram uma alta ductilidade para as misturas contendo PCL-T. Análises de TG mostraram que a temperatura inicial de degradação não variou significativamente com a quantidade de PCL-T. Os espectros de FTIR apresentaram mudanças interessantes com adição de PCL-T que sugerem a existência de interações entre os polímeros. O melhor comportamento durante o processamento dos PHA com adição de PCL-T sugere que a PCL-Ts atuam como platificantes e auxiliares de fluxo, conferindo maior flexibilidade aos materiais biodegradáveis.

A capacidade de B. sacchari acumular poli-3-hidroxibutirato-co-3-hidroxihexanoato (P3HB-co-3HHx) foi confirmada, com até 2 mol% de 3HHx no PHA total (<10% do 3HHx máximo teórico a partir do ácido), indicando flexibilidade da PHA sintase por substratos, porém alta eficiência nas vias catabólicas do hexanoato. Análise da estabilidade térmica do PHA indicou uma temperatura de degradação reduzida, compatível com a presença de unidades 3HHx. Mutantes incapazes de crescer em ácido hexanóico foram obtidos com UV e transposon mini-Tn5, que ainda acumulavam 3HHx a partir de hexanoato mas com redução na capacidade do acúmulo de 3HB e 3HHx. Foram construídos recombinantes abrigando o gene phaB (codificador de 3-cetoacil-CoA redutase) de Ralstonia eutropha ou phaJ1 e phaJ2 (codificadores de enoil-CoA hidratases R-específicas) de Pseudomonas aeruginosa. A expressão de phaB ou phaJ1 aumentou a canalização de 3HB para a PHA sintase, apesar de não aumentar as frações de 3HHx. Monômeros de 3HHx e 3HO foram detectados a partir de ácidos butírico e octanóico, respectivamente. / The ability of B. sacchari to accumulate poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P3HB-co-3HHx) from glucose and hexanoic acid was confirmed. 3HHx content was up to 2 mol% of PHA (<10% of the maximum theoretical 3HHx from the acid), indicating a substrate flexibility of B. sacchari PHA synthase, but high efficiency of hexanoate catabolic pathways. Thermal stability analysis of the copolymer indicated a reduced degradation temperature compatible with 3HHx units. Mutants unable to grow on hexanoic acid were obtained with UV and mini-Tn5 transposon. They still accumulated 3HHx from hexanoate, but the ability to accumulate 3HB and 3HHx was reduced. Recombinants harboring the Ralstonia eutropha phaB (encoding 3-ketoacyl-CoA reductase) and Pseudomonas aeruginosa phaJ1 and phaJ4 genes (encoding R-specific enoyl-CoA hydratases) were constructed. Expression of both phaB and phaJ1 increased the channeling of 3HB to the PHA synthase, despite no increase on 3HHx fraction was observed. 3HHx and 3HO monomers were detected from butyric and octanoic acids, respectively.

A capacidade de B. sacchari acumular poli-3-hidroxibutirato-co-3-hidroxihexanoato (P3HB-co-3HHx) foi confirmada, com até 2 mol% de 3HHx no PHA total (<10% do 3HHx máximo teórico a partir do ácido), indicando flexibilidade da PHA sintase por substratos, porém alta eficiência nas vias catabólicas do hexanoato. Análise da estabilidade térmica do PHA indicou uma temperatura de degradação reduzida, compatível com a presença de unidades 3HHx. Mutantes incapazes de crescer em ácido hexanóico foram obtidos com UV e transposon mini-Tn5, que ainda acumulavam 3HHx a partir de hexanoato mas com redução na capacidade do acúmulo de 3HB e 3HHx. Foram construídos recombinantes abrigando o gene phaB (codificador de 3-cetoacil-CoA redutase) de Ralstonia eutropha ou phaJ1 e phaJ2 (codificadores de enoil-CoA hidratases R-específicas) de Pseudomonas aeruginosa. A expressão de phaB ou phaJ1 aumentou a canalização de 3HB para a PHA sintase, apesar de não aumentar as frações de 3HHx. Monômeros de 3HHx e 3HO foram detectados a partir de ácidos butírico e octanóico, respectivamente. / The ability of B. sacchari to accumulate poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P3HB-co-3HHx) from glucose and hexanoic acid was confirmed. 3HHx content was up to 2 mol% of PHA (<10% of the maximum theoretical 3HHx from the acid), indicating a substrate flexibility of B. sacchari PHA synthase, but high efficiency of hexanoate catabolic pathways. Thermal stability analysis of the copolymer indicated a reduced degradation temperature compatible with 3HHx units. Mutants unable to grow on hexanoic acid were obtained with UV and mini-Tn5 transposon. They still accumulated 3HHx from hexanoate, but the ability to accumulate 3HB and 3HHx was reduced. Recombinants harboring the Ralstonia eutropha phaB (encoding 3-ketoacyl-CoA reductase) and Pseudomonas aeruginosa phaJ1 and phaJ4 genes (encoding R-specific enoyl-CoA hydratases) were constructed. Expression of both phaB and phaJ1 increased the channeling of 3HB to the PHA synthase, despite no increase on 3HHx fraction was observed. 3HHx and 3HO monomers were detected from butyric and octanoic acids, respectively.

Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&amp;D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas. The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells. The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose. / <p>QC 20150508</p>

The aim of this work is to study microbial production of polyhydroxyalkanoates (PHA). Theoretical part is focused on production of PHA using microoganisms and transgenic plants. Bacterial strain Cupriavidus necator H16 was used for laboratory production of PHA. Various waste oils were used as sole carbon and energy source. Salt of propionic adic and 1-propanol were used as intermediate for 3-hydroxyvalerate monomer (3HV) unit. Incorporation of 3HV to polymer can improve material features of PHA. The major part of experimental work was focused to study influence of aeration (concentration of dissolved oxygen) to bacterial growth, selected metabolic pathways and formation of PHA. Furthermore, influence of aeration to monomer composition of polymer was evaluated. According to experimental conclusion of this work it was approved dependance between aeration and monomer composition of PHA. Moreover, it was approved that higher concentration of oxygen supports bacterial growth and influences PHA content in cells. In addition, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH inhibits TCA cycle and enhances accumulation of PHA in cells. For this reason, specific enzymatic aktivity of several selected intracelular enzymes were measured, including those enzymes which can generate NADPH.

The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.

Polyhydroxyalkanoates (PHA) are a family of biodegradable polyesters naturally synthesised by some bacteria and archaea. PHA have high industrial value as bioplastics for packaging and biomedical applications. However, their broader use is hindered by high production costs and uncontrolled variation of polymer properties. The extreme halophile Haloferax mediterranei shows bioprocess advantages that can be exploited for the low cost production of the PHA copolymer Poly(3-hydroxbutyrate-co-3-hydroxyvalterate) (PHBV). The focus of this thesis is to identify process variables responsible for the uncontrolled variation of PHA properties in order to progress towards the robust production of PHBV using H. mediterranei. The outcome of the investigation is a novel cultivation strategy for the reliable synthesis of PHBV copolymers with controlled composition and microstructure showing minor differences in material characteristics. Initially, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and for the first time, nitrate. The nitrogen source and concentration influenced PHBV accumulation and variations in polymer composition were observed with ammonium, highlighting the importance of the control of cultivation conditions. Volatile fatty acids (VFA) were found to be a more direct approach to determine PHBV composition and for the first time were used as substrates in H. mediterranei cultures. When the cells were grown in C4:0/C5:0 mixtures, the 3HV fraction in the PHBV was proportional to the percentage of C5:0 in the feed mixture, allowing the synthesis of copolymers with a predefined composition ranging from pure PHB to pure PHV. The cultivation strategy proved effective for the synthesis of HV rich PHBV, which is not easily obtained due to the 3HV precursor toxicity. The polymer microstructure was controlled using different feeding strategies: co-feeding generated random copolymers, while sequential feeding created block and blend copolymers. The synthesis of block copolymers is of interest because the materials show enhanced yield strength and mechanical strength, making such materials more suitable for commodity uses. Bespoke random, block, and blend copolymers with 0−100 mol% 3HV were synthesized and their thermal and mechanical properties studied. Lastly, high temperature cultivation and several surfactants were tested to enhance the production of bespoke PHBV from VFA. PHBV productivity and accumulation was greatly improved in a fed-batch bioreactor fermentation at 37°C with Tween-80 and the maximum PHBV content 58.9% was obtained. The polymers from shake-flasks and from bioreactors showed minor variations in their material properties, demonstrating the scalability and the robustness of the process developed. Further understanding of the different process variables affecting polymer synthesis and composition was gained in this thesis. It is now possible to produce PHBV with controllable composition, microstructure and minor differences in material characteristics. The novel and robust production strategy developed address the bioprocess challenge of minimising the uncontrolled variation of polymer characteristics that is currently hindering the wider use of PHA hence allowing the production of high quality polymers for commodity goods, packaging and biomedical applications.

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