To mitigate the problems of depleting and soaring price of fossil fuels, the production and use of renewable energy have been vigorously promoted. In Europe, the role of biologically-derived fuels and in particular biodiesel is gradually increasing in prominent. Rapeseed biodiesel is the most widely produced in Europe. As a consequence, enormous amount of by-products from production processes are being generated. Current strategies for managing these by-products (mainly rapeseed meal and crude glycerol) seem not to be economically sustainable. More efficient utilisation could add more value to the production chain which in turn would raise the competitiveness of biodiesel compared to petro-diesel. The aim of the project reported in this thesis was to study the feasibility of producing a value added product, polyhydroxybutyrate (PHB), from by-products generated from rapeseed biodiesel production processes as well as to investigate the effects of methanol, a major impurity in crude glycerol, on growth of Cupriavidus necator, a PHB-producing micro-organism.The preliminary study of C. necator growth in crude glycerol based media revealed that optimum concentration of crude glycerol was in a range 15-25 g/L. It was also found that slight changes in the carbon to nitrogen ratio of the feedstock did not significantly affect the growth while methanol at concentrations beyond 10 g/L did. A model based on a saturation equation was developed and used to successfully predict the inhibition of growth by methanol. From the developed model, mechanisms of the inhibition were proposed. The model could also be used to predict satisfactorily growth or productivity rates in other systems containing short-chain alcohols. The growth in solutions derived from rapeseed meal (designated as hydrolysate) via solid-state fermentation by Aspergillus oryzae followed by hydrolysis of the fermented solids was also studied. The biomass production was found to increase as a function of initial free amino nitrogen (FAN) concentration presented in the hydrolysate. However, at higher initial FAN concentrations, a lower conversion of nitrogen to biomass was observed. PHB production was studied using a feedstock which was a mixture of the hydrolysate and crude glycerol. Total biomass concentration reached 28.8 g/L at 120 h with 86% PHB content. PHB productivity and PHB yield on glycerol were 0.21 g/L•h and 0.32 g/g respectively. These results were comparable with those obtained when pure glycerol and synthetic crude glycerol were used, suggesting that, technically, the use of the generic rapeseed- and crude glycerol-based feedstock to produce PHB is feasible.Overall, the feasibility of producing PHB from rapeseed biodiesel by-products has been demonstrated. The satisfactory result leads to the more important outlook that the generic feedstock derived from rapeseed biodiesel by-products has the potential to be used to produce a wide range of products depending on the micro-organism used. Further development of this process to improve nutrient production efficiency as well as product yields and subsequent integration of the process into the biodiesel production process could well be an important contribution in the development of a sustainable biodiesel industry.

Presented work was focused on biodegradability of bacterial natural and modified polyesters and their composites. The first part of the work was focused on study of influence of PHA granules structure on their biodegradability using selected enzymes and influence of physiological conditions on PHA stability. Overall, tested polymer either in crytalinne or amorphous phase seems to be rezistent to attack of seleced hydrolytic enzymes such as lipases or proteases and is stable in simulated physiological fluids as well. Because of thies results, it is possible to use tested PHA materials in biomedical applications requiring rather resistant biomaterials. Second part of the work was focused on microbial degradation of modified PHA materials considering their potential environmental impact. Both mixed thermophillic culture originaly used in wastewater treatment plant and bacterial strain Delftia acidovorans were employed for biodegradation tests. Composites containing chlorine PHB and PHB films modified using plasticizers were tested. Films containing chlorine PHB cause inhibition of biomass growth to both tested cultures. The highest rate of degradation (31%) was observed in presence of bacterial culture with film containing 10% chlorine PHB. The results show that used microbial population is important factor affecting biodegradability.

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.

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.

Polyhydroxyalkanoates (PHAs) are polyesters, produced by many bacteria and some archaea. The most commonly characterised is polyhydroxybutyrate (PHB). Produced when nutrients are growth limiting and carbon available in excess, PHA serves as a carbon-energy storage material and forms generally spherical insoluble inclusions between 50-500nm in diameter in the cytoplasm. The key enzyme for PHA synthesis is the PHA synthase and this enzyme catalyses the polymerisation of (R)-3-hydroxy fatty acids into PHA. PHA synthase remains covalently attached to the growing polyester chain and is displayed on the surface of the PHA granule. Other proteins associated with PHA granules include depolymerases for mobilisation or degradation of granules, regulatory proteins and phasins, proteins that aid in PHA granule stability. PHA bio-beads displaying an IgG binding protein were produced and used to purify IgG from serum demonstrating that the PHA synthase can be engineered to display functional synthase fusion proteins at the PHA granule surface. Correctly folded eukaryotic proteins were also produced and displayed at the PHA granule surface as phasin fusion proteins. Multiple-functionality was also achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology. The production of functional eukaryotic proteins at the PHA bead surface represents a novel in vivo matrix-assisted protein folding system. Protein engineering of PHA granule surface proteins provides a novel molecular tool for the display of antigens for FACS based analysis and offers promising possibilities in the development of future biotechnological production processes. Overall, the results obtained in this study strongly enhance the applied potential of these polyester beads in biotechnology and medicine.

First artificial PHB granules were prepared under the terms of this thesis. The effect of used PHB solvent, ultrasonic bath temperature and time, solvent evaporation temperature and stabilizing agent nature was investigated using dynamic and electrophoretic light scattering. The most proper parameters were demonstrated at samples which were prepared by dissolving of PHB in chloroform, stabilizing with CTAB or lecithin, ultrasonifying at 35 °C followed by chloroform evaporating at 60 °C. Based on ATR-FTIR and Raman spectroscopy results, it was found out that PHB within the artificial granules were in crystalline form. The native PHB granules were isolated from Cupriavidus necator using either lysosyme, deoxyribonuclease and cell disruption by ultrasonification or digestion with alcalase, SDS and EDTA. Granules obtained by both isolation procedures were characterized by ATR-FTIR, Raman spectroscopy, light scattering techniques and DSC. According to the analyses results, the second mentioned procedure turned out to be more effective for obtaining the polymer in amorphous state. Furthermore, the polymer within granules recovered by using this procedure was thermally more stable. Last but not least, the native PHB granules samples were exposed to effect of acetone, lipase and sodium hypochlorite, assuming that polymer crystallinity should be increased by these chemicals in varying degrees. The highest degree of crystallinity was achieved after their treatment with lipase.

In drei inhaltlich abgeschlossen Teilen werden Fragestellungen bearbeitet, die sich mit dem Einsatz von Polyhydroxybutyraten als Scaffoldmaterialien für das Tissue Engioneering von Knochen beschäftigen. Zunächst wird ein Prozess optimiert, in dem mittels Verpressen und Auslösen von Platzhaltern (Porogen) poröse Träger (Scaffolds) aus Poly-3-hydroxybuttersäure (P3HB) sowie aus P3co4HB hergestellt werden. Diese Scaffolds werden in der Folge mechanisch und strukturell charakterisiert, wobei Druckfestigkeit, Dauerfestigkeit und Viskoelastizität untersucht werden. Im Ergebnis finden sich mehrere Kandidaten, die für die weitere Testung im Tierversuch in Frage kommen. Weiter wird das Abbauverhalten von schmelzgeponnenen P3HB-Fäden untersucht. Dabei wird ein beschleunigtes Modellsystem gewählt, das noch möglichst nahe am physiologischen Fall aber ohne biologisch aktive Komponente (zB. Enzyme) definiert wurde. Die Charakterisierung bedient sich hier der Gelpermeationschromatographie (GPC), des gasgestützten Elektronenrastermikroskops (ESEM), der differentiellen Thermoanalyse (DSC) und der Rasterkraftmikroskopie. Als Ergebnis zeichnete sich ab, dass neben der hydrolytischen Degradation im Gegensatz zu PHB mit kleinerer spezifischer Oberfläche bei den Fäden auch Erosion zum Abbau beiträgt. Eine partikuläre Freisetzung wird nicht beobachtet. Im dritten Teil werden textile Scaffolds aus P3HB mit einer künstlichen extrazellulären Matrix aus Chondroitinsulfaten (CS) und Kollagen versehen. Dem CS kann hier ein positiver Einfluss auf die osteogene Differenzierung von humanen mesenchymalen Stammzellen (hMSC) nachgewiesen werden. Dies wird zum einen durch die verstärkte Expression der alkalischen Phosphatase (ALP) sowie durch die Hochregulation von Proteinen ersichtlich, die bei der osteogenen Differenzierung essentiell sind. In wenigen Gene-Arrays lässt sich ebenfalls erkennen, dass die osteogene Differenzierung durch CS positiv beeinflusst wird. Insbesondere frühe Marker wie ZBTB16 und IGFBPs werden hier identifiziert. Basierend auf den Teilergebnissen wird am Ende ein Beitrag geliefert, der das Tissue Engineering insbesondere für überkritische Röhrenknochendefekte als Methode interessant erscheinen lässt. Dabei werden mechanische Lasten durch konventionelle Fixateure aufgenommen und der Defektraum durch den mehrfachen Einsatz von bio-funktionalisierten flachen Scaffolds gefüllt.:1. Vorwort 3 2. Allgemeine Einführung 5 2.1 Der Knochen 5 2.1.1 Die Knochenbildung 5 2.1.2 Zur Anatomie und Physiologie des Knochens 7 2.2 Tissue Engineering 11 2.2.1 Zelltypen für das Tissue Engineering von Knochen 12 2.2.2 Scaffold Design im Tissue Engineering von Knochen 13 2.3 Polyhydroxyalkanoate 13 2.4 Tissue Engineering am Röhrenknochen 16 2.4.1 Poly(3-hydroxybutyrat)-Scaffolds für das Tissue Engineering von Knochenersatz 17 2.4.2 Matrix Engineering 18 2.5 Ziel der Arbeit 19 3. Mechanik poröser PHB-Scaffolds 21 3.1 Einleitung 21 3.2 Materialien und Methoden 23 3.2.1 Polyhydroxybutyrate und Porogene 23 3.2.2 Uniaxiales Heißpressen 24 3.2.3 Mikrographie 26 3.2.4 Dynamische Differenzkalorimetrie (DSC) 26 3.2.5 Mechanische Druckversuche 26 3.2.6 Mikrocomputertomographie (μCT) 27 3.2.7 Zellviabilität auf den Scaffolds 28 3.3 Ergebnisse 29 3.3.1 Mikrographie 29 3.3.2 Mikrocomputertomographie (μCT) 33 3.3.3 Druckversuche 37 3.3.4 Dynamische Differenzkalorimetrie (DSC) 40 3.3.5 Zellviabilität 40 3.4 Diskussion 40 3.5 Schlussfolgernde Zusammenfassung 46 4. Degradation von P3HB-Fasern 47 4.1 Degradation von Polyhydroxyalkanoaten 47 4.2 Materialien und Methoden 49 4.2.1 Herstellung und Vorbehandlung textiler P3HB-Konstrukte 49 4.2.2 Mechanische Prüfung 50 4.2.3 Beschleunigte Degradation 50 4.2.4 Untersuchung der Oberfläche 50 4.2.5 Dynamische Differenzkalorimetrie (DSC) 51 4.2.6 Gel-Permeations-Chromatographie (GPC) 51 4.3 Ergebnisse 52 4.3.1 Mechanische Tests 52 4.3.2 Die Charakterisierung der Oberfläche 52 4.3.3 Thermische Fasereigenschaften.55 4.3.4 Untersuchung der Molekulargewichte in der GPC 58 4.4 Diskussion 60 4.5 Schlussfolgernde Zusammenfassung 64 5. hMSC auf textilen Scaffolds 67 5.1 Einleitung 67 5.2 Material und Methoden 68 5.2.1 Erzeugung der P3HB-Scaffolds 68 5.2.2 Die Immobilisierung der EZM-Komponenten auf den Scaffolds 69 5.2.3 Isolation, Vorkultur, Besiedlung und Kultur der humanen mesenchymalen Vorläuferzellen 69 5.2.4 Kombinierte Bestimmung von ALP, MTT und Proteingehalt 71 5.2.5 Mikroskopische Untersuchungen 72 5.2.6 Nachweis der Kalziummineralisierung 73 5.2.7 Quantitative real time reverse transcribing polymerase chain reaction (rt-PCR) 73 5.2.8 cRNA Microarray-Untersuchung 74 5.2.9 Zusätzliche Experimente 75 5.3 Ergebnisse 76 5.3.1 Vorhergehende Untersuchung 76 5.3.2 Rasterelektronen-Mikroskopie 77 5.3.3 Konfokale Laser-Scanning-Mikroskopie 79 5.3.4 ALP-Aktivität, SDH-Aktivität und Proteingehalt 82 5.3.5 Mineralisierende Kalziumabscheidung 86 5.3.6 rt-PCR 87 5.3.7 cRNA Microarray-Untersuchung 90 5.3.8 Kulturen von hMSC mit Chondroitinsulfat als gelöstem Zusatz 93 5.4 Diskussion 93 5.5 Schlussfolgernde Zusammenfassung 98 6. Zusammenfassung 101