|
|
Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (referee) ; Sedláček, Petr (advisor)
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.
|
|
|
Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (referee) ; Brázda, Václav (advisor)
Epidemiologické studie prokázaly vliv konzumace rostlinných potravin v prevenci široké škály nemocí. Přírodní antioxidanty přítomné v těchto potravinách, mezi nimiž jsou velmi důležité polyfenoly, mohou být zodpovědné za tuto činnost podporující zdraví. Cílem bakalářské práce je ukázat interakci určitých polyfenolů s genetickým materiálem prostřednictvím různých signálních mechanismů, zejména pokud jde o stabilizaci nekanonické struktury DNA G-kvadruplex a poukázat tak na nejselektivnější látku pro inhibici biochemických procesy. Dále práce obsahuje podrobné informace, které mohou pomoci pochopit, jak mohou polyfenolové sloučeniny interagovat s DNA prostřednictvím epigenetických mechanismů a G4 struktur, a které faktory mohou ovlivnit jejich účinnost. Různé experimenty, biologickým a experimentálním opakováním, byly použity k potvrzení interakce mezi sloučeninami a DNA.
|
|
|
Analytical methods for determination of polyhydroxyalcanoates content in bacterial cells
Černayová, Diana ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with methods that are used for determination of polyhydroxyalkanoates (PHA) in cells of bacteria Cupriavidus necator H16. To the applied methods belong Fourier transform infrared spectroscopy, Raman spectoscopy, turbidimetry and thermal analysis (thermogravimetric analysis and differential scanning calorimetry). The results of each method were compared to amount of PHA determinated by gas chromatography. Fourier transform infrared spectroscopy and Raman spectroscopy revealed a linear relation between ratio of characteristic peaks and amount of PHA. FTIR enabled to determine not only the quantitative amount of PHA, but the quality of the sample (cristallinity, amorphous state) as well. Another method was turbidimetry, which has shown correlation between dispersion of UV-VIS light to amount of PHA in bacterial cells. Thermogravimetric analysis was the most effective technique for determination of PHA. The dependence of weight loss of PHA on its content (found by gas chromatography) was determined with the highest accuracy to gas chromatography. Tha last method- DSC was not alternative replacement for quantitative PHA determination. However, DSC indicated crystalline and amorphous state of the polymer.
|
|
|
Production of polyhydroxyalkanoates by bacterium Schlegelella thermodepolymerans
Černý, Vojtěch ; Černayová, Diana (referee) ; Pernicová, Iva (advisor)
The aim of this work was to study capability of various strains of thermophilic bacteria Schlegelella thermodepolymerans incorporate diverse monomer units to structure of polyhydroxyalkanoates (PHA). Precursors for production mcl-PHA, 4-hydroxybutyrate and 3-hydroxyvalerate were tested. The theoretical part deals with characteristic of studied polymers, properties and applications and usage of thermophilic bacteria in industry. In experimental part there were undergone cultivations with different precursors, which were added in different times and concentrations. Four bacterial strains S. thermodepolymerans from various public collections of microorganisms were used during these cultivations. Levulinic acid, added at start of cultivation, was chosen as the best precursor. Afterwards there was cultivation with concentration line to find out, whether concentration influence composition of copolymer. Increasing the amount of precursor led to decreasing quantity of biomass and PHA, but there were very interesting materials with high amount of 3HV. Addition 8 g/l of precursor led up to 52 %. Subsequently there was found out molecular height of polymers by size-excluion chromatography (SEC) combined with multi-angle light scattering (MALS).
|
|
|
Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (referee) ; Brázda, Václav (advisor)
Epidemiologické studie prokázaly vliv konzumace rostlinných potravin v prevenci široké škály nemocí. Přírodní antioxidanty přítomné v těchto potravinách, mezi nimiž jsou velmi důležité polyfenoly, mohou být zodpovědné za tuto činnost podporující zdraví. Cílem bakalářské práce je ukázat interakci určitých polyfenolů s genetickým materiálem prostřednictvím různých signálních mechanismů, zejména pokud jde o stabilizaci nekanonické struktury DNA G-kvadruplex a poukázat tak na nejselektivnější látku pro inhibici biochemických procesy. Dále práce obsahuje podrobné informace, které mohou pomoci pochopit, jak mohou polyfenolové sloučeniny interagovat s DNA prostřednictvím epigenetických mechanismů a G4 struktur, a které faktory mohou ovlivnit jejich účinnost. Různé experimenty, biologickým a experimentálním opakováním, byly použity k potvrzení interakce mezi sloučeninami a DNA.
|
|
|
Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (referee) ; Sedláček, Petr (advisor)
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.
|
|
|
Analytical methods for determination of polyhydroxyalcanoates content in bacterial cells
Černayová, Diana ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with methods that are used for determination of polyhydroxyalkanoates (PHA) in cells of bacteria Cupriavidus necator H16. To the applied methods belong Fourier transform infrared spectroscopy, Raman spectoscopy, turbidimetry and thermal analysis (thermogravimetric analysis and differential scanning calorimetry). The results of each method were compared to amount of PHA determinated by gas chromatography. Fourier transform infrared spectroscopy and Raman spectroscopy revealed a linear relation between ratio of characteristic peaks and amount of PHA. FTIR enabled to determine not only the quantitative amount of PHA, but the quality of the sample (cristallinity, amorphous state) as well. Another method was turbidimetry, which has shown correlation between dispersion of UV-VIS light to amount of PHA in bacterial cells. Thermogravimetric analysis was the most effective technique for determination of PHA. The dependence of weight loss of PHA on its content (found by gas chromatography) was determined with the highest accuracy to gas chromatography. Tha last method- DSC was not alternative replacement for quantitative PHA determination. However, DSC indicated crystalline and amorphous state of the polymer.
|
guest ::
