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Study on metabolism of polyhydroxyalkanoates in Rhodospirillum rubrum
Rubanová, Blanka ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoate (PHA) metabolism in Rhodospirillum rubrum under different cultivation conditions. The conditions differed in the aeration rate (aerobic, anaerobic, microaerobic) and in the presence of a light source (light cultivation, dark cultivation). First, different volumes of medium were suggested and tested for the fastest and the most suitable inoculum cultivation. Subsequently, cultures of Rhodospirillum rubrum under different conditions in terms of PHA content were compared by gas chromatography. In addition to gas chromatography, other complementary methods were used for better characterization of bacterial growth and metabolism, such as flow cytometry, UV-VIS spectroscopy (transmission and diffusion transmission modes). Fluorescence microscopy and cryo-scanning electron microscopy were used in terms of cell morphology. Furthermore, the ability of bacteria to synthesize color pigments (carotenoids and bacteriochlorophyll a) was investigated. The bacteria were cultivated on different types of media (liquid, solid), the color pigments were subsequently extracted, and their concentrations were calculated. UV-VIS spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) were also used to measure color pigments.
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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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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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Biotechnological production of PHA from waste substrates
Krejčiřík, Roman ; Hrabalová, Vendula (referee) ; Obruča, Stanislav (advisor)
The presented bachelor thesis deals with the study of polyhydroxyalkanoates using wine grape pomace extract. The thesis aims to determine whether it is possible to use wine grape pomace extract as a substrate for polyhydroxyalkanoates (PHA) production. For this research, four thermophilic bacterial strains were selected such as Schlegelella thermodepolymerans, Caldimonas meghalayensis, natural isolate labeled as 34, and Tepidimonas taiwanensis, from which the most suitable bacterium was subsequently selected. The best transformation of residual sugars showed T. taiwanensis, and therefore it was used for all the further fermentation. The extracts were prepared out of wine marc of Veltlínské červené rané, Müller Thurgau, Pálava, Sauvignon, Rulandské bílé, Frankovka (rosé), Frankovka (red wine). The highest PHA concentration was detected after fermentation with concentrated extracts of Sauvignon and Rulandské bílé. Fermentation processes in other extracts were most likely inhibited by the components they contained. Optimization was performed by dilution of extracts with water. The highest concentrations of PHA while using dilute extracts were achieved with the extract from different kinds of Frankovka (rosé) and Frankovka (red wine).
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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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Application of thermal analysis in physiological characterization of microorganisms
Kočiová, Silvia ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the applicability of thermal analysis in the influence of stress factors on microorganisms. The main aim of the presented work was to design and implement a series of experiments to thest the usability of advanced methods of thermal analysis in the characterization of microorganisms. Two bacterial strains were used as model organisms: Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, which differs in the ability to produce polyhydroxyalkanoates. The basic technique used thermogravimetric analysis (TGA), which was optimized and applied in the study of water transport, behavior and determination of intracellular water content with respect to the influence of the presence of intracellular PHB granules. The results of this method were subsequently supported by TEM photos.
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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 bioactive wound dressings and testing their interaction with human cells
Bendová, Agáta ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
The thesis was focused on the preparation and optimization of the preparation of wound dressing from materials with bioactive ingredients. In this work were prepared nanofiber dressings based on polyhydroxybutyrate and non-fibrous dressings from alginate and chitosan. Nanofibers were prepared by electrospinning and forcespinning methods. The bioactive components, which were used to functionalize the prepared dressings, were plant extracts, clotrimazole, ampicillin, lysozyme, and proteolytic enzymes. The theoretical part is focused on the description of the use of nanofibrous and non-fibrous materials in medicine, characterization of materials for the production of wound dressings and bioactive components. Furthermore, this section describes the methods used to prepare and characterize wound dressings. In the practical part were prepared aqueous and oil extracts from selected plants. Extracts were characterized for polyphenols content and antioxidant activity. PHB-based nanofibers were prepared using electrospinning and forcepinning methods. Nanofibers were enriched with selected plant oil extracts and clotrimazol. Modified nanofibres were detemined for antioxidant activity, short-term and long-term stability. Non-fibrous wound dressings were prepared from alginate and chitosan. These dressings were functionalized by the addition of selected aqueous extracts, ampicillin, lysozyme, papain, bromelain, and collagenase. Non-fibrous wound dressings were determined for antioxidant activity, short-term stability and proteolytic activity. The prepared wound dressings were tested for their antimicrobial effects on cultures of Micrococcus luteus, Serratia marcescens, Staphylococcus epidermidis and Escherichia coli. In conclusion, successfully prepared bioactive wound dressings with antioxidant and antimicrobial agents were tested for safety on human cells. The determination was performed using the MTT cytotoxicity test on human keratinocytes.
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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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Preparation and characterization of modern wound covers
Balášová, Patricie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.
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