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Analysis of bacrerial cells employing flow cytometry and flurescence microscopy
Müllerová, Lucie ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.
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Study on resistance of bacteria to selected stress factors
Miléřová, Miluše ; Samek,, Ota (referee) ; Obruča, Stanislav (advisor)
The aim of the master thesis was to study the effect of the accumulation of polyhydroxyalkanoates (PHA) for bacterial resistance to selected stress factors. In the theoretical part the selected stress factors, polyhydroxyalkanoates and the involvement of polyhydroxyalkanoates into stress response of bacteria were reviewed. In the experimental part we used bacteria Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 unable of polyhydroxybutyrate (PHB) accumulation. The resistance of above-mentioned bacterial strains against thermal and osmotic stress was tested. According to the results of the experiment, when the bacteria were exposed to three different concentrations of NaCl (50, 100 and 200 g/l) PHB accumulating strain showed a higer resistance to hyperosmotic stress than the strain unable of PHB accumulation. There was demonstrated with Raman spectroscopy that in the hyperosmotic environment induced crystallization of the intracellular PHB granules. Transmission electron microscopy indicated that strain Cupriavidus necator H16/PHB-4 is subject to plasmolysis during hyperosmotic stress. As a consequence the hyperosmomotic stress occurs to the aggregation intracellular PHB granules in strain Cupriavidus necator H16 but there is no plasmolysis or is much less intensive.
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Methods for identification of PHA producing bacteria
Skřivanová, Veronika ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.
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Controlled Production and Degradation of Selected Biomaterials
Obruča, Stanislav ; Demnerová, Kateřina (referee) ; Vávrová, Milada (referee) ; Němec, Miroslav (referee) ; Márová, Ivana (advisor)
Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.
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Utilization of lignocellulose materials for biotechnological production of polyhydroxyalkanoates
Kučera, Dan ; Márová, Ivana (referee) ; Obruča, Stanislav (advisor)
Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.
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Study of biodegradation of poly(hydroxy alkanoates).
Wurstová, Agáta ; Přikryl, Radek (referee) ; Obruča, Stanislav (advisor)
The master‘s thesis is focused on the study of biodegradation of polyhydroxyalkanoates, namely polymer polyhydroxybutyrate. The first part of the thesis is focused on the study of biodegradation of polyhydroxybutyrate in the form of crystalline granules of PHB and PHB films using selected species of microorganisms from bacteria, yeasts and fungi. As a representative of bacteria was chosen microorganism Delftia acidorovans, as yeast was selected Aureobasidium pullulans and Aspergillus fumigatus as fungi. PHB depolymerase activity was measured employing turbidemtiric method with suspension of PHB granules as substrate. The results showed that D. acidorovans can partially degrade PHB. On the contrary A. pullulans cannot effectively degrade PHB. The most significant degradation ability revealed A. fumigatus, which was able to degrade PHB completely. Extracellular enzymes excreted by these microorganisms when cultivated on PHB materials as sole carbon sources were analyzed by SDS-PAGE. The second part of the thesis deals with the biodegradation of PHB in the form of PHB film, PHB hardened foil and PHB Nanoul fabric using standard composting test. Semi-solid cultivation showed positive results. In the interval from 14 days to two months were all forms of the PHB completely biodegraded. With semi-solid cultivation was also studied biodegradation rate of the polyurethane elastomeric films which were modified by partial replacement of polyester polyol by PHB. The test samples were prepared using PHB from Sigma and the PHB samples prepared at the Faculty of chemistry VUT. Samples with different concentrations of the dispersed PHB (1 %, 5 % and 10 %) in the polyurethane were also object of the study. At the end of the cultivation (after 2 months) were measured mechanical properties in tension of the material, then efficiency of biodegradation by gravimetric analysis and modification of the material surface by microscopic analysis.
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Characterization of specific proteins form selected animal products.
Janhuba, Filip ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.
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Influence of selected parameters on biotechnological production of polyhydroxyalkanoates
Eremka, Libor ; Pekař, Miloslav (referee) ; Obruča, Stanislav (advisor)
The aim of this work is to study microbial production of polyhydroxyalkanoates (PHA). Theoretical part is focused on production of PHA using microoganisms and transgenic plants. Bacterial strain Cupriavidus necator H16 was used for laboratory production of PHA. Various waste oils were used as sole carbon and energy source. Salt of propionic adic and 1-propanol were used as intermediate for 3-hydroxyvalerate monomer (3HV) unit. Incorporation of 3HV to polymer can improve material features of PHA. The major part of experimental work was focused to study influence of aeration (concentration of dissolved oxygen) to bacterial growth, selected metabolic pathways and formation of PHA. Furthermore, influence of aeration to monomer composition of polymer was evaluated. According to experimental conclusion of this work it was approved dependance between aeration and monomer composition of PHA. Moreover, it was approved that higher concentration of oxygen supports bacterial growth and influences PHA content in cells. In addition, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH inhibits TCA cycle and enhances accumulation of PHA in cells. For this reason, specific enzymatic aktivity of several selected intracelular enzymes were measured, including those enzymes which can generate NADPH.
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Biodegradability of bacterial natural and modified polyesters and their composites
Pala, Martin ; Babák, Libor (referee) ; Obruča, Stanislav (advisor)
Presented work was focused on biodegradability of bacterial natural and modified polyesters and their composites. The first part of the work was focused on study of influence of PHA granules structure on their biodegradability using selected enzymes and influence of physiological conditions on PHA stability. Overall, tested polymer either in crytalinne or amorphous phase seems to be rezistent to attack of seleced hydrolytic enzymes such as lipases or proteases and is stable in simulated physiological fluids as well. Because of thies results, it is possible to use tested PHA materials in biomedical applications requiring rather resistant biomaterials. Second part of the work was focused on microbial degradation of modified PHA materials considering their potential environmental impact. Both mixed thermophillic culture originaly used in wastewater treatment plant and bacterial strain Delftia acidovorans were employed for biodegradation tests. Composites containing chlorine PHB and PHB films modified using plasticizers were tested. Films containing chlorine PHB cause inhibition of biomass growth to both tested cultures. The highest rate of degradation (31%) was observed in presence of bacterial culture with film containing 10% chlorine PHB. The results show that used microbial population is important factor affecting biodegradability.
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Possibilities of encapsulation of particular types of macromolecules and bacteria
Kapar, Jiří ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on testing encapsulation methods of enzymes and probiotic bacteria. In the theoretical part a summary of different encapsulation techniques used in food industry is given. Further, materials for encapsulation, above all polysaccharides are presented. Next, some procedures of encapsulation of biopolymers and microorganisms – mainly enzymes and probiotic cultures are discussed. In the experimental part methods for preparation of several types of particles based on polysaccharides and liposomes are introduced. Particles were used for encapsulation of selected hydrolytic enzymes and probiotic strains Bifidobacterium breve a Lactobacillus acidophilus. The encapsulation effectiveness was evaluated by analysis of total proteins and enzyme activities. Particles sizes and their stability in water, in selected model foods and model body fluids were observed, too. According to results obtained in this work it was found that encapsulation of enzymes into polysaccharide particles were succesfull in all types of particles (encapsulation effectivness was more than 50 %). Polysaccharide particles showed a very good stability in body fluids as well as in model foods. As the most suitable materials for enzymes encapsulation chitosan and liposomes were found. Polysaccharide particles were used also for the encapsulation of microorganisms. The stability of particles with lactic acid bacteria was similar to particles containig enzymes, very good stability was verified aslo in model foods and model body fluids. Encapsulation enables long-term stabilization of biologically active compounds as well as posibility of their transport and controlled releasing in gastrointestinal tract. Encapsulation of probiotic bacteria could preserve their viability and long-term survival until the product expiration date. Thus, encapsulation is one of the most promissing procedures for production of foods and food suplements of great quality and high additional value.
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