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Optimisation of Isolation Procedure and Characterization of Amorphous PHB granules
Kratochvíl, Zdeněk ; Obruča, Stanislav (referee) ; Kalina, Michal (advisor)
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.
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Preparation of nanoparticles and nanofibers with antimicrobial components
Sosková, Simona ; Veselá, Mária (referee) ; Skoumalová, Petra (advisor)
The presented diploma thesis is focused on the preparation of new materials with antimicrobial effect. Liposomes and nanofibers from polyhydroxybutyrate containing clotrimazole and natural extracts with good antifungal and antioxidant effects were prepared. The theoretical part contains examples and short description of using nanoparticles and nanofibers in cosmetics and medicine and the description of plants which have positive and potential antimycotic effects. Moreover, methods for particles and fibers characterisation were shortly described. In the experimental part, natural water and lipid extracts were prepared and spectrophotometrically characterised for the content of polyphenols, flavonoids and the antioxidant activity. Liposomes and liposomes containtng PHB were prepared from selected extracts and the encapsulation effectivity, shortterm and longterm stability via determination of polyphenols were determined. Prepared particles were characterized with DLS method (size) and zeta- potential (stability). PHB nanofibers functionalised with selected lipid extracts and clotrimazole were prepared via electrospinning and forcespinning, and examined via FLIM and FTIR-ATR methods and spectrophotometry was used for antioxidant activity and release of active substances determination. Antifungal properties of prepared particles, extracts and fibers using the test system Candida glabrata were studied. Finally, cytotoxicity of selected samples was tested with MTT assay using human keratinocytes.
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Polyhydroxyalkanoates and their role in bacterial biofilms
Rucká, Markéta ; Samek,, Ota (referee) ; Obruča, Stanislav (advisor)
This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.
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Utilization of spent wood chips for biotechnological production of PHA
Ladický, Peter ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the suitability of wood shavings and sawdust as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most appropriate approach of hydrolysis of wood shaving and sawdust and the effect of polyphenol and furfural concentration on ability to accumulate PHA was studied. Burkholderia sacchari had greater ability to accumulate PHA compared to Burkholderia cepacia. PHB values 87–89 % were achieved when Bulkholderia sacchari was cultivated on medium that contained detoxified hydrolysate of sawdust. Sawdust is therefore a promising substrate for microbial production of PHA in terms of reducing production costs and high content of PHB in biomass.
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Controlled biotechnological production of polyhydroxyalkanoates
Šnajdar, Ondřej ; Pekař, Miloslav (referee) ; Obruča, Stanislav (advisor)
Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.
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Proposal of systems for targeted transoprt of compounds of sweet taste
Demová, Radoslava ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on possibility of immobilization sweeteners on polymeric system. In the theoretical part information about sweeteners, carrier systems and the possibility of immobilization technique were reviewed. In the experimental part preparation of nanoparticles of polyhydroxybutyrate (PHB) was optimized. The PHB particles were characterized by dynamic light scattering (DLS). PHB particles were activated by plasma treatment. The surface morphology was studied using infrared and Raman spectroscopy. Immobilization of following substances with a sweet taste was tested: glucose, fructose, sucrose, mannitol, sorbitol and erythritol. From these compounds only sucrose was immobilized on PHB particles successfully. Prepared immobilized particles were exposed to the artificial stomach juice, intestinal juices and bile acids and amount of released sucrose was monitored. Finally, long-term stability prepared particles was measured and also the amount of sucrose released was determined.
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Properties of biocomposites based on flax fibers
Pelánová, Markéta ; Obruča, Stanislav (referee) ; Přikryl, Radek (advisor)
This thesis is focused on composites, which are composed of degradable components. The theoretical part deals with composite materials, treatment of fibers and properties of composites. The purpose of this study is to create a composite consisting of polyhydroxybutyrate, which is reinforced with flax fibers with various modifications. The thesis also evaluates the effect of reinforcing fibers in the composite material on the tensile properties and hydrolytic stability and degradability of the composite in compost in laboratory conditions.
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Biotechnological production of polyhydroxyalkanoates using wastes of coffee production
Vašíčková, Monika ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of production of polyhydroxyalkanoates (PHA) by bacteria Burkholderia cepacia and Burkholderia sacchari. Production of PHA has been studied using spent coffee grounds hydrolysates which have been prepared by different aproaches. In the thesis is compared standard method of coffee ground hydrolysis by mineral acid followed by enzymatic hydrolysis. Hydrolysates which have been produced were analyzed in terms of concentration of saccharides and efficiency of hydrolysis. The best producer of PHA (based on results) was bacterium Burkholderia cepacia, in the medium which has been prepared by hydrolysis of spent coffee grounds extracted by 60% solution of ethanol. Biomass yield of this cultivation was 3,553 g/l with 32,472% PHA content. This PHA contained 6,09% 3-hydroxyvalerate. In the other experiment, we verified alternative hydrolysis of spent coffee grounds – by commercially available enzymes. We used cellulase, hemicellulase and the enzymatic cocktail which has been produced by mould. Hemicellulase was the most effective hydrolytic enzyme and its application resulted in production of the highest amount of biomass – 5,708 g/l. In this cultivation, only homopolymer PHB has been which is probably caused by the fact, that during the enzymatic hydrolysis levulinic acid or any other potential precursor of 3HV is formed.
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Controlled production of polyhydroxyalcanoates by bacterial strain Ralstonia eutropha using different substrates
Šnajdar, Ondřej ; Duroňová, Kateřina (referee) ; Márová, Ivana (advisor)
This bachelor’s thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Ralstonia eutropha. Production of PHA on different substrates has been studied to lower the costs of feedstock. In theoretical part the review has been done about the most important types of PHA, production strains and possibilities of cultivation of Ralstonia eutropha using different substrates. In practical part there has been studied production of PHA on different vegetable oils, including waste oils from different sources (restaurants, homes, food companies). Incorporation of different precursors for copolymer production control was studied too. The highest yields of poly-3-hydroxybutyrate (P3HB) have been achieved using waste oils. For economical and ecological reasons these oils are very suitable substrates. Using concentration 20 g/l of waste rapeseed oil from university canteen there has been produced 13,32 g/l of biomass containing 58,63% of P3HB in 84th hour of cultivation. The highest yield of PHA in precursors study has been achieved applying 1% propanol in 24th hour of cultivation. The PHA increase has been 97%. This PHA was composed from 91% of 3HB and 9% of 3-hydroxyvalerate.
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Modification of polyurethane by biodegradable poly(hydroxybutyrate)
Kupka, Vojtěch ; Žídek, Jan (referee) ; Vojtová, Lucy (advisor)
Cílem předložené bakalářské práce byla syntéza elastomerního polyuretanu plněného biodegradabilním polyhydroxybutyrátem. Vzorky byly podrobeny analýze morfologie pomocí rastrovací elektronové mikroskopie, mechanické vlastnosti byly testovány jednoosou tahovou zkouškou. Práce se taktéž zabývá předpovědí mechanických vlastností na základě teorií kaučukové elasticity. Bylo zjištěno, že výsledný materiál je částicový kompozit, kde se vzrůstajícím hmotnostním obsahem plniva v materiálu vzrůstá modul pružnosti. Predikce tahových křivek pomocí softwaru SEC.exe ukazovala pro reálný průběh křivky nereálné hodnoty hustot polymerních řetězců. U modelů, které vykazovaly reálné hodnoty hustot, byly naopak hodnoty napětí vyšší, než ve skutečnosti naměřené.
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