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Adaptation to adverse osmotic conditions as a tool for evolutionary enginnering of bacteria
Drotárová, Lenka ; Nováčková, Ivana (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with the application of osmotic stress as a tool for evolutionary engineering of PHA producing bacterial strains. The aim of this thesis is to evaluate a bacterial adaptation to hypoosmotic environment, as an engineering tool in order to increase the production of PHA. The theoretical part focuses on the evolutionary engineering principle, methods of the strategy and the effect of physical factors on microorganism. The aim of experimental part was to performed an adaptive evolutionary experiment with the bacterial strain Halomonas halophila CCM 3662. Reduced osmotic pressure was used as a stressing factor during the serial cultivation. In order to generate PHA producing mutant strains, each passage was characterized using spectrophotometric and gravimetric method and by GC-FID. It was found that after the long-term cultivation, the mutant strain HH35, cultivated in 35 g/l NaCl, was associated with the highest biomass and PHB concentration. The 15th and 30th passages, along with the wild type strain H. halophila were subjected to further cell-robustness analysis with the application of hyper- and hypoosmotic shock. The stress response, viability of cells and morphological changes were analyzed using FC and TGA methods. Isolated polymers were characterized using FTIR analysis.
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Preparation of biodegradable films from PHA copolymers and evaluation of their application potential
Drotárová, Lenka ; Přikryl, Radek (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified.
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Preparation of biodegradable films from PHA copolymers and evaluation of their application potential
Drotárová, Lenka ; Přikryl, Radek (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified.
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Adaptation to adverse osmotic conditions as a tool for evolutionary enginnering of bacteria
Drotárová, Lenka ; Nováčková, Ivana (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with the application of osmotic stress as a tool for evolutionary engineering of PHA producing bacterial strains. The aim of this thesis is to evaluate a bacterial adaptation to hypoosmotic environment, as an engineering tool in order to increase the production of PHA. The theoretical part focuses on the evolutionary engineering principle, methods of the strategy and the effect of physical factors on microorganism. The aim of experimental part was to performed an adaptive evolutionary experiment with the bacterial strain Halomonas halophila CCM 3662. Reduced osmotic pressure was used as a stressing factor during the serial cultivation. In order to generate PHA producing mutant strains, each passage was characterized using spectrophotometric and gravimetric method and by GC-FID. It was found that after the long-term cultivation, the mutant strain HH35, cultivated in 35 g/l NaCl, was associated with the highest biomass and PHB concentration. The 15th and 30th passages, along with the wild type strain H. halophila were subjected to further cell-robustness analysis with the application of hyper- and hypoosmotic shock. The stress response, viability of cells and morphological changes were analyzed using FC and TGA methods. Isolated polymers were characterized using FTIR analysis.
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