Bio-plastics have emerged as a promising alternative to conventional petrochemical derived plastics particularly over the past few decades. Numerous production methods for bio-plastics have been researched; however, work remains towards developing a commercially viable and economical process. The purpose of this research was to develop a sustainable fermentation strategy for production and scale-up of medium-chain length polyhydroxyalkanoates (mcl-PHAs), or bio-plastics, using a novel strain of the gram negative bacterium Pseudomonas putida, LS46, with biodiesel derived waste glycerol (WG) as feedstock. Experiments were conducted to gain a basic understanding of the general growth patterns exhibited by LS46. Thereafter, flask-batch experiments were conducted to study effects of variation in media conditions upon cell biomass production and mcl-PHA accumulation. Subsequently, optimal medium conditions observed within flasks were scaled-up and employed in the operation of a pilot-scale fermenter to increase production capacity for mcl-PHAs. It was concluded that mcl-PHA production at commercial levels could be viable with advanced process optimization. / October 2015

Polyhydroxyalkanoates (PHAs) are biodegradable plastic synthesized by microorganisms from renewable carbon resources and they are promising substitutes for conventional fossil-fuel-based plastics due to their similar physical properties. Pure cultures of particular microorganisms are commonly used for industrial PHA production but high production costs due to requirements of sterile conditions and refined substrates hinder the mass production of PHAs. Thus, model development for PHA production by microbes is essential to investigate the PHA formation and microbial metabolisms for enhanced productivity and PHA contents. In the present study, a comprehensive numerical model has been developed and calibrated for the non-growth associated PHA production process by Cupriavidus necator. The model parameters were calibrated with 8 selected experimental studies and the simulation results show good agreement with experimental data. Two methods were used to conduct sensitivity analysis: the simple method and the overall relative sensitivity analysis method. Maximum specific residual biomass growth rate was the most sensitive parameter. The calibrated model was used to investigate fed-batch feeding strategies that optimize PHA accumulation by limited nutrient feeding in the PHA production phase. The simulation results showed limited phosphorous feeding accumulated more PHA than limited nitrogen feeding. The optimal feeding strategy was determined to be limited phosphorous feeding at 5% of initial phosphorous during the PHB production phase, yielding simulated 226.0 g/L PHB at the end of the 168-hour operation. / Thesis / Master of Applied Science (MASc)

The aim of this diploma thesis is the preparation of liposome nanoparticles enriched with PHA and PHA nanofibers. The nanostructures served to encapsulate extracts of lipophilic and hydrophilic nature. The characterization of the properties of nanostructures such as polydispersity, size, colloidal stability, long-term stability after exposure to various environments such as seawater, water from the Brno dam and tap water, and finally the cytotoxicity of fibers with extracts was addressed. The theoretical part is focused on different types of water, human skin, coffee and subsequently carotenoids. It continues by describing of possibilities of extraction and preparation of lipophilic and hydrophilic extracts and possibilities of their determination is discussed. Finally, it describes the possibilities of preparation and characterization of PHA based nanomaterials. The practical part deals with the preparation of liposome particles and fibers enriched with PHA with encapsulated extracts, their characterization, and their subsequent exposure to various environments. Monitoring of their long-term stability was carried out, but the release of the encapsulated extracts into the environment to which the nanoparticles and nanofibers were exposed was also measured. Finally, the interaction of nanofibers with live HaCaT cells was monitored, and cytotoxicity assays determined the viability of the cells after interaction with the nanofibers.

Τα τελευταία χρόνια σημαντική ερευνητική δραστηριότητα έχει επικεντρωθεί στην παραγωγή βιοδιασπώμενων βιοπολυμερών, ικανών να αντικαταστήσουν τα συμβατικά πλαστικά, από ανανεώσιμες πηγές. Μεταξύ αυτών, ιδιαίτερο ενδιαφέρον έχει προκύψει στο πεδίο παραγωγής πολυυδροξυαλκανοϊκών οξέων (PolyHydroxyAlkanoates, ΡΗΑs) από διαφορετικούς τύπους αποβλήτων, χρησιμοποιώντας είτε καθαρές είτε μικτές μικροβιακές καλλιέργειες. Προκειμένου να γίνει επιλογή μικτών καλλιεργειών απαιτείται ο πρότερος εγκλιματισμός και εμπλουτισμός τους με τη χρήση κάποιου είδους περιορισμού των θρεπτικών συστατικών σε συστήματα περιοδικής λειτουργίας, ενώ είναι επίσης σύνηθες να περιλαμβάνεται ένα πρώτο βήμα οξίνισης των αποβλήτων πριν από την αξιοποίησή τους για παραγωγή των βιοπολυμερών. Στα πλαίσια της παρούσας εργασίας, αναπτύχθηκε μια μικτή εμπλουτισμένη καλλιέργεια από εδαφόβιους μικροοργανισμούς, χρησιμοποιώντας αντιδραστήρα άντλησης-πλήρωσης (Draw-Fill) σε συνθήκες περιορισμού αζώτου. Ως πηγή άνθρακα χρησιμοποιήθηκε μίγμα των πτητικών λιπαρών οξέων οξικό, βουτυρικό και προπιονικό σε αναλογία 1:1:1. Η εγκλιματισμένη και εμπλουτισμένη μικροβιακή καλλιέργεια χρησιμοποιήθηκε στη συνέχεια για τον εμβολιασμό καλλιεργειών σε αντιδραστήρα διαδοχικών παρτίδων (SBR) αλλά και άντλησης-πλήρωσης, προκειμένου να διερευνηθεί η δυνατότητα παραγωγής PHAs υπό περιορισμό βασικού θρεπτικού (οργανικό άζωτο) αλλά και υπό διαφορετικής πηγής άνθρακα, όπως: α) μίγματα οξέων, β) καθαρή γλυκερόλη, γ) καθαρή 1,3 προπανοδιόλη δ) μίγματα γλυκερόλης και οξέων και ε) οξινισμένη βιομηχανική γλυκερόλη. Η οξινισμένη γλυκερόλη είναι ουσιαστικά το ζυμωτικό μίγμα που παράγεται κατά την αναερόβια ζύμωση της γλυκερόλης και αποτελείται από υπολειμματική γλυκερόλη που δεν έχει υποστεί ζύμωση, 1,3 προπανοδιόλη, πτητικά λιπαρά οξέα και αιθανόλη. Τα παραγόμενα PHAs ανακτήθηκαν σε όλες τις περιπτώσεις υπό την μορφή λεπτού υμενίου με τη χρήση χλωροφορμίου και έγινε υπολογισμός των αποδόσεων. Πραγματοποιήθηκε περαιτέρω ανάλυσή των ιδιοτήτων των PHAs ως προς τη δομή, τις θερμοδυναμικές και φυσικοχημικές τους ιδιότητες καθώς και ως προς τις μηχανικές τους σε επιλεγμένες περιπτώσεις. Σε όλες τις περιπτώσεις εκτός αυτής της καθαρής γλυκερόλης, τα προϊόντα ήταν συμπολυμερή, αποτελούμενα κατά κύριο λόγο από PHB, PHV και PHHx, με μοριακά βάρη μεταξύ 76,7.104–181,6.104 Da. Οι υψηλότερες αποδόσεις, από την άποψη της ικανότητας συσσώρευσης, παρατηρήθηκαν για τις τροφοδοσίες οξέων και γλυκερόλης που έχει υποστεί ζύμωση, φθάνοντας τα 0,6±0,04g PHA/g TSS και 0,41±0,04g PHA/g TSS αντίστοιχα, ενώ η υψηλότερη παραγωγικότητα επιτεύχθηκε στην περίπτωση μικτής τροφοδοσίας (οξέα/γλυκερόλη), φθάνοντας σε 0,39±0,01g PHA/g COD που καταναλώνεται. / In recent years a lot of research has been focused on the production of biodegradable bioplastics from renewable resources, in order to replace the conventional plastics. Among them great interest has been shown on the production of Polyhydroxyalkanoates (PHAs) from different types of wastes, using either pure or mixed bacterial cultures. The latter usually requires that microbial mixed cultures have been previously acclimated and enriched using some kind of nutrient limitation under periodic feeding, whereas it is also common to include a first step of acidification of the waste prior to PHAs production. In the present study, a mixed microbial culture derived from soil was developed, using nitrogen limitation in a draw-and-fill reactor (DFR). The carbon source was a mixture of acetate, butyrate and propionate at ratio 1:1:1. The acclimated culture was then used as inoculums in Sequencing Batch Reactors (SBR) and DFRs in order to produce PHAs under nutrient limitation and different kinds of carbon sources such as a) acids mixture, b) pure glycerol, c) pure 1.3 propanediol d) mixture of glycerol and acids and e) anaerobically fermented waste glycerol (acidified industrial glycerol). The latter consisted from residual glycerol, acids, 1.3 propanediol and ethanol. The produced bioplastics were recovered in all cases in the form of films using chloroform as extractant, and PHAs yields were estimated. PHAs films were further analyzed in terms of their chemical structure, thermal properties and in selected cases, their mechanical properties. It was shown that in all cases, but for pure glycerol, co-polymers consisting mainly from PHB, PHV and PHH were obtained, with molecular weighs ranging from 76.7.104–181.6.104Da. The highest yields in terms of accumulation capacity were observed for acids and fermented glycerol as carbon source reached 0,6±0,04g PHA/g TSS and 0.41±0.04g PHA/g TSS, whereas the highest productivity was achieved for mixed acids/glycerol as carbon source reached 0.39±0.01g PHA/g COD consumed.

The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.

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.

Foi avaliado o potencial de Halomonas sp. HG01, uma bactéria halófila, isolada de uma lagoa salina no Peru, com relação à produção de polihidroxialcanoatos (PHA). Halomonas sp. HG01 foi capaz de utilizar eficientemente sacarose, glicose e frutose como fontes de carbono. Os melhores desempenhos foram obtidos com glicose ou sacarose. 3-Hidroxibutirato (3HB) foi o único monômero detectado a partir de carboidratos. Precursores de outros monômeros foram avaliados. Monômeros de 3-hidroxivalerato (3HV) foram incorporados ao polímero quando os ácidos propiônico ou valérico foram supridos. 4-Hidroxibutirato (4HB) também foi utilizado como monômero pela PHA sintase de Halomonas sp. HG01. Engenharia evolutiva permitiu aumentar a eficiência de consumo de xilose e glicerol. Cultivos em biorreator permitiram atingir produtividades volumétricas de 0,13; 0,07 e 0,06 g/L.h, quando glicose, xilose e glicerol, respectivamente, foram supridas. Maiores produtividades deverão ser atingidas com aumento na densidade celular dos cultivos. / The potential of Halomonas sp. HG01, a halophilic bacteria isolated from a saline lagoon in Peru, was evaluated regarding the production of polyhydroxyalkanoates (PHA). Halomonas sp. HG01 was able to use efficiently sucrose, glucose and fructose as carbon sources. The best performance was obtained with glucose or sucrose. 3-hydroxybutyrate (3HB) was the only monomer detected from carbohydrates. Precursors of other monomers were evaluated. 3-Hydroxyvalerate (3HV) was inserted in the polymer when propionic or valeric acids were supplied. 4-Hydroxybutirate (4HB) was also used as a monomer for the PHA synthase from Halomonas sp. HG01. Evolutive engineering allowed to increase the uptake efficiency of xylose and glycerol. Cultures in bioreactor allowed achieving volumetric productivities of 0.13; 0.07 and 0.06 g /L.h when glucose, xylose, and glycerol, respectively, were supplied. Higher productivities should be achieved with increased cell density of cultures.

Les poly (3-hydroxyalcanoate)s (PHAs) constituent une solution alternative aux plastiques issus des ressources pétrolières en raison de leur biodégradabilité et leur biocompatibilité. Cependant, les coûts de production élevés et les difficultés de mise en œuvre des PHAs ont limité leur développement à plus grande échelle. Il convient donc de modifier les PHAs afin d’accroître leurs propriétés et de développer des stratégies permettant de réduire leurs coûts de production pour permettre leur utilisation en remplacement des plastiques conventionnels. Parmi les matières premières issues des ressources renouvelables, les huiles métropolitaines de colza ou de tournesol sont des candidats intéressants pour la synthèse et la modification chimique des PHAs de par leur coût compétitif, leur biodisponibilité et leurs fonctionnalités intrinsèques. Ainsi, notre travail a porté sur réduction des coûts de production des PHAs en utilisant des substrats tels que l’huile de colza ou le glycérol. La souche sélectionnée, Haloferax mediterranei, a démontré sa capacité à biosynthétiser du PHB92HV8. Par ailleurs, nous avons développé deux approches permettant d’améliorer les performances des PHAs : la plastification par des molécules terpéniques issues des plantes et la synthèse de réseaux semi-interpénétrés (semi-IPNs) par réaction de thiolène entre l’huile de tournesol et un thiol trifonctionnel au sein d’une matrice de PHAs linéaire. L’utilisation de terpènes pour la formulation des PHAs a permis de réduire la température de mise en œuvre du polymère de 7 °C et d’augmenter sa souplesse. La synthèse d’un réseau semi-interpénétré biosourcé a permis d’améliorer la stabilité thermique des PHAs et d’augmenter leur allongement à la rupture de 2400 %. Enfin, de nouveaux matériaux biosourcés ont également été produits à partir de terpènes et d’huiles végétales, en faisant appel à un procédé simple et vert. Les matériaux obtenus, aux propriétés intéressantes en termes de flexibilité et d’élasticité ont la capacité de piéger et de libérer des molécules hydrophobes telle que la molécule d’eugénol aux propriétés antibactériennes et antifongiques. Ainsi, une large gamme de bioplastiques a été synthétisée en valorisant les huiles végétales et les PHAs, dont les propriétés variées pourraient concurrencer les plastiques actuels issus des ressources fossiles / Poly (3-hydroxyalkanoate)s (PHAs) are an alternative to petroleum-based plastics because of their biodegradability and their biocompatibility. However, the high production costs, the limited mechanical performance and the narrow processing window of PHAs have limited their development on a larger scale. It is therefore necessary to modify the PHAs in order to increase their properties and develop strategies to reduce their production costs to allow their use as replacement for conventional plastics. Among the raw materials derived from renewable resources, metropolitan rapeseed or sunflower oils are interesting candidates for the synthesis and chemical modification of PHAs because of their competitive cost, their bioavailability and their built-in functionalities. Thus, we aimed to reduce the cost productions of PHAs by using rapeseed oil and glycerol as cheap substrates. The strain, Haloferax mediterranei, has demonstrated its ability to biosynthesize a PHB92HV8. In addition, we have developed two approaches to improve the performance of PHAs: plasticization of PHAs by terpene molecules from plants and synthesis of semi-interpenetrating networks (semi-IPNs). The use of terpenes for the formulation of PHAs reduced the processing temperature of the polymer and increased its flexibility. The synthesis of a biobased semi-IPN is obtained by crosslinking sunflower oil and a trifunctional thiol, using the thiolene reaction, within a matrix of linear PHAs. The network improved the thermal stability of PHAs and increased their elongation at break of 2400%. Finally, new biobased materials were also produced from terpenes and vegetable oil, using a simple and "green" process. The resulting materials exhibited flexibility and elasticity with the ability to absorb and to release antibacterial and antifungal hydrophobic molecules such as the eugenol. Therefore, a wide range of bioplastics have been synthesized using vegetable oils, PHAs or a combination of both, with wide range of properties to compete with plastics derived from fossil resources

This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.

Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.