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Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (oponent) ; Sedláček, Petr (vedoucí práce)
The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.
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Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (oponent) ; Sedláček, Petr (vedoucí práce)
The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.
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