Metody kvalitativní a kvantitativní analýzy PHA v buňkách cyanobakterií
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
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.
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.
Description
Keywords
Polyhydroxyalkanoáty cyanobaktérie, Synechocystis sp. PCC 6803, Synechocystis salina CCALA 192, GC–FID, FT-IR, turbidimetria, integračná guľa, UV-VIS spektrometria, termogravimetria, Polyhydroxyalkanoates, cyanobacteria, Synechocystis sp. PCC 6803, Synechocystis salina CCALA 192, GC–FID, FT-IR, turbidimetry, integration sphere, UV-VIS spectroscopy, thermogravimetry analysis
Citation
ČERNAYOVÁ, D. Metody kvalitativní a kvantitativní analýzy PHA v buňkách cyanobakterií [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2020.
Document type
Document version
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Language of document
sk
Study field
Chemie pro medicínské aplikace
Comittee
prof. Ing. Miloslav Pekař, CSc. (předseda)
prof. RNDr. Ivana Márová, CSc. (místopředseda)
doc. Ing. Pavel Diviš, Ph.D. (člen)
doc. Ing. Petr Dzik, Ph.D. (člen)
doc. Ing. Stanislav Obruča, Ph.D. (člen)
prof. RNDr. Zbyněk Zdráhal, Dr. (člen)
Date of acceptance
2020-09-04
Defence
Obhajoba proběhla podle následujícího schématu: prezentace studentky-vyjádření vedoucí/ho-oponentský posudek-reakce na posudek-diskuse s komisí. Studentka přednesla výborný výtah výsledků své diplomové práce, řádně zodpověděla všechny dotazy oponentské i členů komise, pohotově reagovala na připomínky. V diskusi tak studentka prokázala výbornou schopnost orientace v teoretických i praktických základech problematiky diplomové práce. Komise zhodnotila její diplomovou práci celkově jako výbornou.
Otázky vznesené během diskuse:
Dzik: Můžete popsat rozdíly mezi měřením přímé a difůzní transmitance? Jaké benefity přináší měření difůzní reflektance?
Márová: Prosím vysvětlete rozdíly mezi metodami použitými pro stanovení PHB.
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení