Analytical methods for determination of polyhydroxyalcanoates content in bacterial cells

Táto bakalárska práca sa zaoberá metódami na stanovenie polyhydroxyalkanoátov (PHA) v bunkách baktérie Cupriavidus necator H16. Medzi použité metódy patrí infračervnená spektroskopia s Fourierovou transformáciou (FTIR), Ramanova spektroskopia, turbidimetria a termické analýzy (termogravimetrická analýza a diferenčná skenovacia kalorimetria). Výsledky z metód boli porovnávané s obsahom PHA stanoveného plynovou chromatografiou. Pri infračervenej spektroskopií s Fourierovou transformáciou a Ramanovej spektroskopií sa zistila pozitívna korelácia pomeru charakteristických pásov k obsahu PHA. S použitím FTIR bolo možné okrem kvantitatívneho stanovenia PHA zistiť aj kvalitu vzorky (kryštalickosť /amorfnosť). Ďalej sa použila turbidimetrická analýza, pri ktorej sa sledovala závislosť rozptylu UV-VIS žiarenia na obsahu PHA v bakteriálnych bunkách. Najefektívnejšou technikou sa ukázala byť termogravimetrická analýza. Závislosť úbytku hmotnosti v oblasti spaľovania PHA, na jeho obsahu (zisteného plynovou chromatografiou) bola stanovená s najvyššou koreláciou k plynovej chromatografií. Metóda DSC sa neukázala byť vhodnou alternatívnou náhradou na stanovenie obsahu PHA, ale umožnila stanoviť či sa polymér nachádza v stave kryštalickom, alebo amorfnom.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.

The economic capital determination in non-life insurance

This paper takes an overall look at the stochastic model used for computing the solvency capital requirement in non-life insurance within the scope of Solvency II. Its purpose is to investigate the methods of aggregation of the risks from the various lines of business, especially the method of multivariate Archimedean copulas

Faktory ovplyvňujúce vstup podnikateľského subjektu na zahraničné trhy

The diploma thesis focuses on determining the critical success factors of SMEs breweries penetration of the Lithuanian market. The first part deals with the terminology related to the SMEs, the internacionalization process itself, situation analysis, the management decision process and last but not least, risk analysis. The practical part is devoted to to the analysis and comparison of slovak and lithuanian market environment through PESTEL and Porter analysis, a selection of the location and a form of penetration of the Lithuanian market. Morover, it specifies risks and critical success factors of the internationalization of the company using a map of risks and a sensitivity analysis. The overall topic is complemented of a business plan, its prediction and SWOT analysis

The economic capital determination in non-life insurance

This paper takes an overall look at the stochastic model used for computing the solvency capital requirement in non-life insurance within the scope of Solvency II. Its purpose is to investigate the methods of aggregation of the risks from the various lines of business, especially the method of multivariate Archimedean copulas

Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria

Táto diplomová práca sa venuje overeniu využiteľnosti vybraných fyzikálne-chemických a spektroskopických metód pri charkterizácií cyanobaktérií, a to sa so zvláštnym dôrazom na možnosti kvalitatívnej a kvantitatívnej analýzy polyhydroxyalkanoátov (konkrétne polyhydroxybutyrátu (PHB)) akumulovaných v bunkách cyanobaktérií. Vzorkovú základňu práce tvorili kultúry cyanobakterálnych kmeňov Synechocystis sp. PCC 6803 a Synechocystis salina CCALA 192, ktoré boli kultivované viacerými spôsobmi za účelom pokrytia čo najširšieho spektra fyziologických podmienok a obsahov PHB a to konkrétne pri využití autotrofného spôsobu kultivácie na trepačke a v multikultivátori v základnom kultivačnom médiu, v médiách obohatených o 2% soli (NaCl) a tiež mixotrofných kultiváciách– médiách s rôznymi typmi uhlíkového substrátu. Po niekoľko týždňovej kultivácií boli získané cyanobakteriálne kultúry komplexne analyzované nasledujúcimi technikami– bunkové suspenzie boli analyzvané prietokovou cytometriou a UV-VIS spketrofotometriou (v transmisnom a difúznom transmisnom móde), zatiaľ čo suchá biomasa bola charakterizovaná plynovou chromatografiou, ktorou bol stanovený presný obsah PHB, a ďalej FT-IR spektrometrickou a termogravimetrickou analýzou. Cieľom práce bolo jednak posúdiť, či niektorá z uvedených metód môže predstavovať rýchlu a dostupnú alternatívu stanovenia obsahu PHB k najčastejšie využívanej metóde plynovej chromatografii, ale tiež posúdiť, aké ďalšie informácie o fyziologickom stave cyanobaktériálnych buniek môžu testované metódy poskytnúť. Najvyššia korelácia na obsahu PHB bola stanovená pre parametre stanovené infračervenou spektroskopiou, pri ktorej boli dôležité konkrétne píky z charakteristických vlnočtov pre polyhydroxybutyrát. Slabé korelácie na obsahu PHB boli dosiahnuté pri termogravimetrickej analýze a cytometrii, kedy bola využitá hydrofóbna fluorescenčná sonda BODIPY 439/503, ktorá sa naviazala na lipofilné časti buniek. Popri stanoveniu PHB bolo možné prietokovou cytometriou a UV-VIS spektrometriou s difúznym transmisným módom stanoviť pigmenty, ktoré sa nachádzajú v siniciach (ako sú chlorofyl, fykocyanin a karotenoidy). Na záver boli výsledky experimentov zo všetkých techník porovnané PCA analýzou, aby sa zistila vzájomná podobnosť všetkých analyzovaných vzoriek.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.