No description available.

Thermoplastic blends of several biodegradable polymers with lignin (L) and lignin esters were prepared by solvent casting and melt processing. Among the biodegradable thermoplastics were cellulose acetate butyrate (CAB), poly-hydroxybutyrate (PHB), poly-hydroxybutyrate-co-valerate (PHBV), and a starch-caprolactone blend (SCL). Lignin esters included acetate (LA), butyrate (LB), hexanoate (LH), and laurate (LL). Blend characteristics were analyzed in terms of thermal and mechanical properties. The results indicate widely different levels of interaction between two polymer constituents. Melt blended samples of CAB/LA and CAB/LB were compatible on a 15-30 nm scale when probed by dynamic mechanical thermal analysis, and the glass transition temperatures of the blends followed Fox equation, whereas those of CAB/LH and CAB/LL showed distinct broad transitions on the same scale. Melt blending produced well dispersed phases whereas large phase separation evolved out of solvent castings. Crystallinity and melting points of PHB and PHBV were affected by the incorporation of lignin component, revealing some interaction between the blend constituents. Blends of SCL with L and LB revealed significant effect on crystallinity and melting temperatures of poly-caprolactone component, revealing polymer-polymer interaction between SCL and lignin components. An increased degree of crystallinity was observed in the case of higher-Tg L compared to lower Tg LB. Improvememt in modulus (and in some cases strength also) was observed in almost all blends types due to the glassy reinforcing behavior of lignin. / Master of Science

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate that has been extensively studied as a potential biodegradable replacement for petrochemically derived plastics. The synthesis pathway of PHB is native to Ralstonia eutropha, but the genes for the PHB pathway have successfully been introduced into Escherichia coli through plasmids such as the pBHR68 plasmid. However, the production of PHB needs to be more cost-effective before it can be commercially produced. A mathematical model for PHB synthesis was developed to identify target genes that could be genetically engineered to increase PHB production. The major metabolic pathways included in the model were glycolysis, acetyl coenzyme A (acetyl-CoA) synthesis, tricarboxylic acid (TCA) cycle, glyoxylate bypass, and PHB synthesis. Each reaction in the selected metabolic pathways was modeled using the kinetic mechanism identified for the associated enzyme. The promoters and transcription factors for each enzyme were incorporated into the model. The model was validated through comparison with other published models and experimental PHB production data. The predictive model identified 16 enzymes as having no effect on PHB production, 5 enzymes with a slight effect on PHB production, and 9 enzymes with large effects on PHB production. Decreasing the substrate affinity of the enzyme citrate synthase resulted in the largest increase in PHB synthesis. The second largest increase was observed from lowering the substrate affinity of glyceraldehyde-3-phosphate dehydrogenase. The predictive model also indicated that increasing the activity of the lac promoter in the pBHR68 plasmid resulted in the largest increase in the rate of PHB production. The predictive model successfully identified two genes and one promoter as targets for genetic engineering to create an optimized strain of E. coli for PHB production. The substrate-binding sites for the genes gltA (citrate synthase) and gapA (glyceraldehyde-3-phosphate dehydrogenase) should be genetically engineered to be less effective at binding the substrates. The lac promoter in the pBHR68 plasmid should be genetically engineered to more closely match the consensus sequence for binding to RNA polymerase. The model predicts that an optimized strain of E. coli for PHB production could be achieved by genetically altering gltA, gapA, and the lac promoter.

No description available.

L’accumulation de biopolymère de réserve (PolyHydroxyAlcanoates ou PHA) par la souche Cupriavidus necator, à partir de substrats de type acides gras volatils (acide butyrique, acide propionique et acide acétique) a été étudiée. Elle est induite par une limitation phosphore. Les performances atteintes lors des cultures se situent parmi les meilleures de la littérature pour ce type de substrat : jusqu’à 66 g.L-1 de biomasse totale avec un pourcentage d’accumulation massique de 88% en PHB –PolyHydroxyButyrate- ou en PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- comportant jusqu’à 52% de motifs d’HV.Pour chaque source carbonée, une caractérisation cinétique et stœchiométrique de la souche a été réalisée en l’absence d’effets inhibiteurs dus aux substrats acides grâce à des cultures de type Fed-Batch avec des apports non limitants et non inhibiteurs en carbone. Il a été dégagé :- un taux de croissance maximal de la souche de 0,33 h-1 pour les trois acides étudiés- une relation entre vitesse spécifique de production de PHA et taux de croissance fixée par la disponibilité et les flux de production de NADPH2 avec un découplage inverse pour les taux de croissance supérieurs à 0,05 h-1 et un couplage partiel pour les taux de croissance inférieurs- un optimum de 0,35 Cmole.Cmole-1.h-1, associé à un taux de croissance de l’ordre de 0,05 h-1.- une amélioration de la production de PHB en termes de vitesses spécifiques mais également en termes de rendements si une faible croissance résiduelle est maintenueLa réponse de la souche à un excès de substrat acide a été caractérisée via l’étude de régimes transitoires induits par des pulses sur des cultures continues préalablement stabilisées en régime permanent. Il a été montré qu’en excès de phosphore, face à un brusque excès de substrat, la souche est incapable d’adapter rapidement son taux de croissance. L’excès est donc dirigé vers la production de PHA dont les voies sont plus rapidement mobilisables. En conditions limitantes de phosphore, le substrat excédentaire est utilisé pour la production de PHA. L’inhibition par les acides se traduit par une diminution des capacités de biosynthèse de la biomasse et des PHA entrainant une réduction de l’assimilation du carbone puis une diminution des rendements de conversion. D’autre part la sensibilité d’un système continu à un excès de substrat dépend du point de fonctionnement choisi : plus il est optimal en termes de vitesse, moins le système est robuste. L’acide propionique est très inhibiteur comparé aux autres acides étudiés (dès 3-4 mM contre 30-40 mM). Il n’agit pas simplement via une accumulation excessive dans le cytoplasme mais il exerce également une inhibition spécifique des voies métaboliques.Un antagonisme entre les substrats (acide acétique et butyrique) a été constaté et expliqué grâce à une analyse des flux métaboliques. L’acide acétique est assimilé préférentiellement pour produire la biomasse, l’énergie et les cofacteurs nécessaires à la production de PHA, alors que l’acide butyrique est utilisé pour la synthèse de PHB. La proportion maximale d’acide acétique admise dans l’alimentation en fonction des conditions fixées en régime permanent est calculée et peut être limitée à 40% du carbone.Enfin il a été déterminé que si une croissance résiduelle est assurée grâce à un apport en phosphore, le pourcentage maximal d’HV dans le polymère dépend du taux d’acide propionique dans l’alimentation et ne peux dépasser 33 ± 5% sur acide propionique pur. Par contre, si aucune croissance résiduelle n’est assurée, il est possible de convertir l’acide propionique en motifs d’HV uniquement / Reserve Biopolymer (PolyHydroxyAlkanoates or PHA) accumulation by the strain Cupriavidus necator, from Volatile Fatty Acids (VFA, like butyric acid, propionic acid and acetic acid) was investigated. This production is induced by a phosphorus limitation. For this type of substrates, performances reached during cultures are among the best listed in the literature: up to 66 g.L-1 of total biomass with 88% (w/w) of PHB –PolyHydroxyButyrate- or PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- with a HV content up to 52 Mole%.For each carbon source, kinetic and stoechiometric characterization has been carried out thanks to Fed-Batch cultures with non-limiting and non-inhibitory carbon feed. It has been established:- a maximal growth rate of 0,33 h-1 for the three acid investigated- a relationship between specific PHA production rate and growth rate which is set by the availability and production flux of NADPH2. For growth rate above 0,05 h-1, there is an inverse coupling. For growth rate under 0,05 h-1, there is a partial coupling.- an optimum of 0,35 Cmole.Cmole-1.h-1 is associated with a growth rate of 0,05 h-1.- if a low residual growth rate is maintained, an improvement of PHB production is recorded in terms of specific production rate and yieldsThe response of the strain to an excess of acid substrate was characterized through the investigation of transient state induced by pulsed addition of substrate during continuous cultures stabilized in steady state. It was shown that in excess of phosphorus, when there is a substrate excess, the strain is unable to quickly adapt its growth rate, so the excess is directed to PHA production whose ways seem to be more easily mobilized. Under phosphorus limitation, an excess of substrate is used for PHA production. Acid inhibition results in a decrease in biomass and PHA production capacity which leads to a decrease in carbon assimilation and conversion yields. The sensitivity of a continuous system to an excess of substrate depends on the chosen operating point: the more it is optimal in terms of specific production rate, the less the system is robust. Propionic acid is highly inhibitory compared to the other acids studied (from 3-4 mM versus 30-40 mM). It does not act only via an excessive accumulation in the cytoplasm but also exerts a specific inhibition of metabolic pathways.An antagonism between substrates (acetic and butyric acid) has been established and explained thanks to the Metabolic Flux Analysis. Acetic acid is preferentially used to produce biomass, energy and cofactors for PHA synthesis, whereas butyric acid is used to product PHB. According to the conditions set during steady state, maximal content of acetic acid admitted in the feed can be calculated. It can be limited to 40% of the carbon in the feed.Finally if a growth rate is maintained thanks to a phosphorus supply, the maximal HV content in polymer is function of propionic acid in the feed and cannot exceed 33 ± 5 Mole% on pure propionic acid. Conversely, if there is no residual growth, a total conversion of propionic acid into HV is allowed

Des produits de consommation de première nécessité aux produits les plus fortuits, l'emballage, en particulier plastique, constitue aujourd'hui un élément indispensable de notre vie quotidienne. Son utilisation intensive dans le domaine agroalimentaire pour un usage unique à courte durée de vie incite aujourd'hui à s'orienter vers de nouveaux matériaux d'origine renouvelable et biodégradables, aux caractéristiques similaires que leurs homologues issus des ressources fossiles. Les mélanges à base de biopolymères et de biopolyesters peuvent être une bonne alternative. Dans ce présent travail de thèse, des mélanges de farine de blé, thermoplastifiée par du glycérol et de l'eau, et des polyesters biosourcés et biodégradables tels que le PLA et/ou le PHB ont été obtenus par extrusion bivis et moulés par injection thermoplastique. Les caractéristiques thermiques, thermomécaniques dynamiques, morphologiques, mécaniques et barrières de ces nouveaux matériaux ont été étudiés. De l'acide citrique a été utilisé comme agent de compatibilisant pour améliorer l'interface amidon/PLA. Les différentes investigations nous ont permis de mettre au point différents types de formulations aux caractéristiques mécaniques et barrières à la vapeur d'eau intéressantes pour la fabrication de corps de barquettes d'emballage alimentaire de denrées périssables comme la viande ou les fromages. L'aptitude au contact alimentaire des matériaux farine thermoplastifiée/polyester et l'impact de l'incorporation des polyesters sur la biodégradabilité de ces matériaux ont également été étudiés. / From basic and essential to unnecessary and optional consumer products, packaging, particularly plastic, is today an indispensable part of our daily life. Its extensive use in the food industry for a single use and for a short shelf-life encourages us today to move towards new renewable and biodegradable materials with similar characteristics than their counterparts from fossil resources. Biopolymers and biopolyesters blends can be a good alternative. Within the framework of this present work, wheat flour, thermoplasticised by glycerol and water, and biobased and biodegradable polyesters such as PLA and/or PHB, were blended using an industrial twin screw extruder and were injection-molded into thermoplastic materials. Thermal, dynamic thermomechanical, morphological, mechanical and barriers properties of these new materials were studied. Citric acid was used as a compatibilizer to improve the interface starch/PLA. The different investigations have allowed us to develop various types of formulations, with mechanical characteristics and barrier properties to water vapor, very attractive for manufacturing plastic food packaging which can be used for meats or cheeses. Food contact suitability and biodegradability of thermoplasticised wheat flour/polyester materials have also studied.

Made available in DSpace on 2015-03-26T13:46:31Z (GMT). No. of bitstreams: 1 texto completo.pdf: 893904 bytes, checksum: 428b03ed61d5a4d87eed9198cfbc34b9 (MD5) Previous issue date: 2007-10-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Biomaterials used today to treat bones' defects and fractures still bear some deficiencies to be corrected. This study is a preliminary analysis of composites' made of hydroxyapatite (HAP-91®) and polyhydroxybutyrate (PHB) essentials characteristics to be used in orthopedics. The composites were studied in osseous defects concerning their biocompatibility, osseointegration, osseoconduction, and biodegradation. Bone defects were made bilaterally in the olecranon of 30 white rabbits and each filled with a composite, with a total of 12 members per group. Limbs in the 1 group received composite # 1: 10% of HAP-91® and 90% of PHB; limbs in 2 group received composite # 2: 25% HAP- 91® and 75% PHB; and in 3 group the composite # 3: 50% of each biomaterial. Defects in the control group were not filled. Limbs were evaluated in the first eight days after surgery and again at 45 and 90 days afterwards for lameness, circumference, pain, dehiscence, and infection. Radiographs were taken 8, 45 and 90 days after surgeries when four animals in each group were euthanized and samples with defects taken for histology. Significant differences in circumference data were found for all groups before and one day after surgery, what did not happen with four and eight days. No significant differences were found between groups for circumference as well as for the other clinical parameters. Radiographs taken 8 days after surgery showed no bone- composite apposition, but those for the 45th and 90th days after showed that composites were in direct contact with bone. Histological observations indicated a normal repair process in the 8th day, i.e., a vascularized, dense connective tissue and new, early trabecular bone; this evolved to fully organized trabecular bone and the lateral cortical completely formed by the 90th-day. At the three observation dates, tissues in 1, 2, and 3 group samples were found similar to those in control, where more bone than soft tissues were found in the defect. Projections of connective and bone tissues were seen inside composites #2 and 3. No inflammatory cells in any place whatsoever were found at any observation date. Clearly more bone built up in 3 samples, and at its interface, than in the 1 and 2 groups samples. At the 45 and 90th days, in all three groups, osteoclasts were seen in the bone-composite interface, and also composite pieces, detached from the original block, were found within the bone structure and surrounding soft tissues. Conclusion is that the composites are biocompatible, osseoconductive, integrate to bone and can undergo in vivo degradation. / Os biomateriais hoje utilizados no tratamento das fraturas ósseas e da perda do tecido ósseo ainda apresentam deficiências a serem corrigidas. O presente trabalho consta da análise preliminar de características essenciais para utilização de compósitos de hidroxiapatita (HAP-91®) e polihidroxibutirato (PHB) em ortopedia. Estudou-se a biocompatibilidade, osseointegração, osseocondução e biodegradação dos compósitos em defeitos ósseos. Foram implantados três compósitos fabricados com diferentes proporções desses biomateriais em defeitos circulares bilaterais no olécrano de 30 coelhos, onde cada membro recebeu um compósito, com um total de 12 membros por grupo. Os membros do grupo 1 receberam compósitos contendo 10% de HAP-91® e 90% de PHB, do grupo 2 contendo 25% de HAP-91® e 75% de PHB, do grupo 3, 50% de cada biomaterial e os animais do grupo controle não receberam compósito. Os membros foram avaliados quanto à claudicação, circunferência do membro, sensibilidade dolorosa, deiscência e infecção nos oito primeiros dias após a cirurgia e aos 45 e 90 dias. Foram radiografados imediatamente após a cirurgia e aos oito, 45 e 90 dias. Nessas mesmas datas, 4 animais de cada grupo foram eutanasiados, coletando-se material para processamento histológico. Existiu diferença significante entre os valores de circunferência do membro no pré-operatório e do primeiro dia após a cirurgia dentro de todos os grupos, o que não ocorreu em relação aos dias quatro e oito. Não houve diferença entre os grupos para esse parâmetro assim como para os demais. Não foi observado contato direto entre osso e compósito nas radiografias obtidas aos oito dias após as cirurgias, mas aos 45 e 90 dias nos três grupos tratados observou-se este contato. À análise histológica do grupo controle observou-se processo típico de reparação, ou seja, tecido conjuntivo denso vascularizado e trabéculas ósseas novas aos oito dias com evolução para osso trabecular mais organizado aos 90 dias e cortical lateral restabelecida. Os tecidos observados nos grupos 1, 2 e 3 foram semelhantes ao controle, sendo a proporção de tecido ósseo formada no defeito, maior do que a de tecidos moles em todas as datas. Nos grupos dois e três foram observadas projeções de tecido ósseo e conjuntivo no interior dos poros do compósito. Infiltrados inflamatórios não foram observados em nenhum momento. O grupo 3 apresentou interface com maior proporção de tecido ósseo formado na região do defeito e no interior do compósito do que os grupos 1 e 2. Observou- se osteoclastos nas bordas dos compósitos e fragmentos dos mesmos separados do bloco principal aos 45 e 90 dias nos três grupos. Concluiu-se que os compósitos são biocompatíveis, osteocondutores, se integram ao tecido ósseo e são degradados in vivo.

The presented diploma thesis deals with the optimalisation of preparation and the preparation of combined nanostructured antimicrobial biomaterials itself. In the theoretical part, a review focused on used materials and consequently preparation of nanoparticles and nanofibers was elaborated. Furthermore, the used antimicrobial substances – curcumin and ampicillin, and the principle of cytotoxicity assay were described.In practical part the optimalisation process is described. Furthermore the safety of prepared materials and used antimicrobial substances on HaCaT cell line was tested, in order to confirm their possible further use in cosmetic and pharmaceutical industry. Great part of the thesis deals with evaluation of the antimicrobial activity of used substances and prepared combined nanomaterials on multiple microorganisms from grampositive bacteria, gramnegative bacteria and yeasts. Also the release speed of antimicrobial substances from prepared nanomaterials was determined by spectrophotometer. The amount of released ampicillin from prepared nanomaterials was determined by liquid chromatography.

The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates (PHA). PHA can be used as biodegradable materials. Bacterial strain Cupriavidus necator was used for laboratory production of PHA. This bacterium was cultivated in medium with various precursors to produce copolymers of 3HB with 3HV or 4HB. Another part of the work was aimed at cultivation of C. necator on different waste substrates, especially oils, with the aim to achieve the highest production of polymer. Another large part of the thesis is dedicated to isolation strategies of PHA using enzymes. Commercially used proteases – alcalase and pancreatin – can be used with advantages for digestion of bacterial cells. A number of optimization experiments showed that application of proteases leads to enhancement of PHA purity to about 13%. Purity increase up to 90 % was achieved by adding a surfactant, which promotes the solubility of non-PHA forming polymer. This surfactant increases the purity of 20 % when compared to control. The last part of presented work deals with the use of enzyme solution isolated from Bacillus subtilis medium. Its application to C. necator culture led to the yield of polymer at a purity exceeding 95 %. These results could represent the basis for new isolation strategies, which can lead to more efficient yield of PHA.

Legionella pneumophila ist ein Gram-negatives, ubiqitär verbreitetes Proteobakterium, das als Auslöser der Legionärskrankheit gilt. Die Ergebnisse dieser Arbeit bestätigen, dass Serin eine wichtige Kohlenstoffquelle für Aminosäuren darstellt und dass L. pneumophila für die Aminosäuren Isoleucin, Leucin, Phenylalanin, Tyrosin, Histidin, Prolin und Valin auxotroph ist. Innerhalb der Replikationsvakuole greift L. pneumophila auf Aminosäuren des Wirts zurück und inkorporiert diese in Proteine. Die untersuchte Spezies besitzt die Fähigkeit zur de novo-Biosynthese von Serin. Die vorliegende Arbeit zeigt zudem erstmals, dass Glukose für die Biosynthese von Aminosäuren sowie Polyhydroxybutyrat (PHB) verwendet wird. Der Entner-Doudoroff-Weg (EDW) kann als Haupt-Katabolismusroute von Glukose identifiziert werden. Ein Deletionsstamm des EDW zeigt gegenüber dem Wildtypstamm in nährstoffarmer Umgebung deutlich verminderte Fitness nach erfolgreicher Replikation innerhalb A. castellanii. Die Glukoamylase GamA wird in dieser Arbeit erstmals als verantwortliches Enzym für die Stärke- und Glykogenhydrolyse von L. pneumophila charakterisiert. Der putative Transkriptionsaktivator YozG bindet sowohl im 5’-DNA-Bereich von gamA als auch im eigenen putativen Promotorbereich und reguliert die Hydrolyseaktivität von GamA. Es werden zudem Argumente für die Hypothese erbracht, dass YozG auch als cis-aktives Element fungiert. Die Biosynthese von Polyhydroxybutyrat (PHB) aus Glukose findet während des spät-exponentiellen Wachstums statt und steigert sich in der stationären Phase. Eine verminderte PHB-Menge im EDW-Deletionsstamm wird als Erklärung für die verminderte Fitness in nährstoffarmer Umgebung diskutiert. Die Ergebnisse dieser Arbeit zeigen, dass L. pneumophila neben Aminosäuren auch Glukose sowie die natürlich vorkommenden Glukosepolymere Glykogen und Stärke als Kohlenstoffquellen zur Biosynthese von Aminosäuren und PHB nutzt und dass diese Synthese zur Fitness der Spezies beiträgt. / Legionella pneumophila is a Gram-negative proteobacterium causing Legionnaires’ disease. The results of this study confirm that serine is a carbon source for amino acids and that L. pneumophila is auxotrophic for the amino acids isoleucine, leucine, phenylalanine, tyrosine, histidine, proline, and valine. L. pneumophila uses amino acids of the host cells to incorporate them into proteins. Serine is synthesized de novo. Furthermore, this work demonstrates for the first time that glucose is used for the biosynthesis of amino acids and polyhydroxybutyrate (PHB). The Entner-Doudoroff pathway is identified as the predominant pathway for the catabolism of glucose. In a nutrient-depleted environment, after replication has taken place in A. castellanii, a deletion strain of the Entner-Doudoroff pathway exhibits strongly reduced fitness as compared to the wildtype. The glucoamylase GamA is characterized as the enzyme responsible for the starch and glycogen degrading activity of L. pneumophila for the first time. The putative transcription activator YozG binds to the 5’ region of gamA as well as to his own putative promoter region and regulates GamA activity. Furthermore, arguments are provided to support the hypothesis that YozG also acts as a cis-active element. The biosynthesis of polyhydroxybutyrate (PHB) from glucose starts in the late exponential phase and increases in the stationary growth phase. As an explanation for reduced fitness of the Entner-Doudoroff deletion strain in nutrient-depleted environment, a reduced amount of PHB is proposed. The results of this work prove that L. pneumophila uses not only amino acids but also glucose and the natural glucose polymers starch and glycogen as carbon sources for the biosynthesis of amino acids and PHB and that thereby the fitness of the species is increased.