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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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Isolation, characterization and application of biomedically important polymer P(3HB-co-4HB)
Krupičková, Kristýna ; Přikryl, Radek (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the isolation and characterization of copolymer P(3HB-co-4HB). The teoretical part was prepared as a literature search which describe polyhydroxyalkanoates in general, their structure, synthesis, degradation and isolation. Furthermore, copolymers containing 4HB units are mentioned in this thesis and there is also no mentioned of the biosynthesis and biodegradation of copolymer P(3HB-co-4HB). The first aim of this diploma thesis was to find out which solvent is the best for copolymer extraction and then characterize obtained copolymer P(3HB-co-4HB). The isolated copolymer was characterized by gas chromatography, SEC-MALS, thermal analysis and SEM. In the second part of the thesis, release of model biologically active substance from the PHA films was studied. Rhodamine 6G dye was selected for the simulation, which was used to stain the copolymer films and the P(3HB) films. It was observed that film prepared from P(3HB-co-4HB) copolymer released entrapped substance much faster than film made from P3HB homopolymer, and, in addition, the copolymer was substantially more susceptible to enzyme degradation.
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Interaction of PHA-based particles and fibres with human cells
Tarageľ, Matej ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
This bachelor thesis deals with the preparation and characterization of PHA based nanoparticles and nanofibers and their subsequent influence on the living human skin cell. Theoretical part is aimed at polyhydroxyalkanoates, preparation of PHA based nanoparticles and nanofibers and methods of their physical, biological and chemical characterization. Further, the theory of liposomes, their clasification, preparation and physical and chemical properties are discussed too. Next, the theoretical part focuses on the cultivation of human cells and general information on selected cell lines. The experimental part is focused on the optimalization of the preparation of liposomes and fibers enriched by PHA. Further, characterization of stability and interactions of these nanomaterials with human HaCaT cells was proved. Cytotoxic and genotoxic tests were performed and determination of concentration of phospholipids in prepared particles was realized too.
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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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Plasticizing of polyhydroxyalkanoates in microbial cells - inspiration for development of artificial controlled release systems
Liczka, Jan ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
The diploma thesis is focused on the study of the mechanism of plasticization of polyhydroxyalkanoates (PHA) in vivo, focusing mainly on methods of isolation native PHA granules and physical and chemical methods of initiation their phase transitions. The literature search prepared in this work deals with this microbial polyester and further focuses on individual methods of isolating PHA from bacterial cells. The main task of the experimental work was to optimize the isolation process of native PHA granules, as well as to design and test processes that initiate the PHA crystallization in isolated granules, as well as analytical procedures to correctly detect this phase transition. Isolation of PHA granules from Cupriavidus necator was performed by enzymes, centrifugation several times and sonication of the cells. Fourier transform infrared spectroscopy (FTIR) and an enzymatic method in which amorphous PHA is selectively degraded by PHA-depolymerase with turbidimetric detection of its loss in dispersion was used to detect PHA crystallization. Techniques based on fluorescence staining of granules (fluorescence spectroscopy and flow cytometry) were used in a more detailed study of the mechanism of PHA plasticization in granules. The results of the analyzes confirmed, among other things, that the optimized procedure makes it possible to isolate granules in the amorphous state, which remain amorphous even after drying and are comparable to native granules in their basic physicochemical properties.
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Use of mealworm (Tenebrio molitor) for isolation of polyhydroxyalkanoates from bacterial biomass and simultaneous production of food
Najbrtová, Johana ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the use of mealworms (Tenebrio molitor) to isolate polyhydroxyalkanoates from bacterial biomass and current food production. Cupravidus necator H16 and Schlegelella thermodepolymerans were used to prepare bacterial biomass containing polyhydroxyalkanoates (PHA). The ability of mealworms to isolate PHA from bacterial biomass of various types was determined. First, the content of PHA and crude proteins in individual types of biomasses was determined. Subsequently, two biological isolation experiments were performed, in both cases the results were compared with a control sample that consumed bran. The conversion of feed into the form of fecal pellets was monitored, where the best results were obtained from a sample of mealworms fed with dried biomass C. necator H16. Furthermore, the weight loss during the experiment, the percentage of survivors, the amount of feed not consumed, and the number of pupae were evaluated. The fecal pellets obtained by biological isolation had a low PHA content and were therefore subjected to purification. The reagent with the best results was 0,1 M NaOH. Another part of this work was to determine the crude protein content of mealworm flour made from mealworms from experiment two. The sample fed with biomass produced by Schlegelella thermodepolymerans had the highest protein content.
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Polyhydroxyalkanoates as metabolites of prokaryotic microorganisms adapted to extreme conditions
Vodička, Juraj ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The objective of this work is a study of polyhydroxyalkanoates as bacterial carbon and energy storage forms, which are produced intracellularly. The theoretical part of the study is focused on physical properties of PHA, their biotechnological production and degradation. The experimental part deals with production of polyhydroxyalkanoates by halophile bacterium Halomonas organivorans using several different carbon substrates. Firstly, the gene encoding for PHA-synthase, phaC, was successfully amplified and detected. Extracellular lipolytical activity was also proved. Secondly, the most appropriate substrate for bacterial growth and PHA production were found to be hydrocarbons, especially galactose and mannose. Due to these results, it is highly desirable to use coffee grounds for the biotechnological production of PHB, as galactomannan-rich waste. On the other hand, lactose and xylose have shown to be unsuitable carbon sources. For that reason, waste whey and lignocellulosic materials rich for pentoses are inconvenient substrates for the intended purpose. There is a potential use of waste molasses because of good results of sucrose at contrast. By the cultivation on kitchen waste and waste glycerol were the growth slow and the production of PHA reduced significantly. The optimal salt concentration for bacterial growth and PHA production were discovered as 60 grams per litre. Addition of several precursors of 3-hydroxyvalerate after 24 hours of cultivation have not induced the copolymer P(3HB-co-3HV) formation. Even though these precursors have inhibited the bacterial growth, the significant increase of the PHB percent contend was observed. In general, the yield of the production has been considerable, it should be noted than almost in each successful cultivation the PHA content in bacterial cells exceeded 50 weight %. With a respect to results, more experiments for full evaluation of H. organivorans as a potential biotechnological PHA producing bacteria are regarded.
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Development of microbioreactor for microbial biotechnology
Jakešová, Michaela ; Szotkowski, Martin (referee) ; Krejčí,, Jan (advisor)
This master thesis deals with the development of a minibioreactor for microbial biotechnologies. The AlgaTox system – an analytical photobioreactor from BVT Technologies – was the default unit of the new device. The working volume of the new minibioreactor is in the range of 4 to 8 ml. The minibioreactor was composed of a minithermostat, a reaction vessel, an oxygen electrode, a temperature and pH probe, accessories for the supply of air to the liquid and an insertion for the transport of liquid from / to the reactor. The functionality of the assembled device and its characteristics were measured. Furthermore, an operating procedure for the decontamination process using a hydrogen peroxide mist was developed for the new equipment. An operating procedure for culturing microorganisms in a minibioreactor was also set up. Pilot cultivations of Halomonas halophila were demonstrated in the prepared equipment. In these cultivations, three assemblies for air supply to the liquid were tested. However, none of the assemblies was able to provide a sufficient supply of oxygen to cell culture – the dissolved oxygen value always dropped to 0%. For the further development, a new assembly was designed for the supply of air to the liquid - an aeration ring from a membrane tube.
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Photoprotective properties of PHB granules
Hesko, Ondrej ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The objective of this work is study and design of physical-chemical methods for verification of photoprotective properties PHB inclusions using bacteria Cupriavidus necator H16 and its mutated bacteria strain Cupriavidus necator PHB-4 which differs in production capability of polyhydroxyalkanoates. Basic information about of polymer inslusions and methods used for proving photoprotectivity are discussed in theoretical part. Methods such as nephelometry and continous fluorescence microphotolysis (CMF), which is used to characterize the mechanism of interaction of light with inclusions, were optimized. In experimental part properties of light scattering in bacteria were determined and characteristic curves were obtained. Further the photostability of fluorescence probe specific for nucleic acids were compared in bacteria strains. This method should prove bigger tolerance of bacteria H16 for UV light. It should also define possible interaction of nucleic acids with PHB inclusions.
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Influence of oxidative pressure on bacterial cells
Dugová, Hana ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the impact of oxidative pressure on the bacterial strain Cupriavidus necator in its two forms: Cupriavidus necator H16 producing PHB granules and Cupriavidus necator PHB-4 as a mutant that is not capable of producing granules. The thesis compares different influences causing oxidative stress that is demonstrated by the occurrence of ROS in the cell. Furthermore, the cells were analysed with a flow cytometer and a fluorescence microscope. During the analysis it was necessary to use different types of fluorescence probes. The oxidative stress was created by means of hydrogen peroxide at various concentrations. Further tests focused on Fenton’s reaction including ammonium iron sulfate and cobalt chloride hexahydrate. Propidium iodide as a fluorescence probe was used to determine the viability of the bacterial cells, and BODIPY was tested as a lipophilic dye. Finally, the ROS in the cell was detected by H2DCFDA and CM–H2DCFDA, fluorescence probes that had to be optimised for the respective technique. The result of this bachelor thesis is the confirmation of the hypothesis that PHA granules production increase the resistance of Cupriavidus necator against oxidative stress.
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