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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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Organical chemicals from biomass
DOLEŽAL, David
This bachelor thesis deals with the processes of biomass as a renewable resource for the production of chemicals that could compete chemicals obtainable from fossil sources. Processing of biomass can be divided into two main approaches namely a thermochemical and biotechnological processing of biomass processes. This processing involves many processes such as hydrolysis, crushing, fermentation, combustion or high temperatures and pressures, and many other thermochemical or biotechnological processes associated with the nature of the structure of the biomass. As the most important products of biomass processing can be considered biofuels represented in my work mainly by ethanol, but also methanol and other organic chemicals such as furfural, levulinic acid and hydroxymethylfurfural derived from the processing of C5 and C6 sugars located in the fundamental structure of biomass. These organic chemicals can also serve as a chemical platform and subsequent modifications of them can get products with the same or very similar properties of the products of the petrochemical industry.
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