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Encapsulation of soil bacteria in hydrogel carriers
Orišková, Sofia ; Smilek, Jiří (referee) ; Sedláček, Petr (advisor)
The goal of this bachelor thesis was both to review existing literature regarding the topic of inoculation of soil bacteria and test a relevant encapsulation method and optimize it. The evaluation process involved the study of various encapsulation techniques that involve hydrogel carriers suitable for agronomic purposes. Encapsulation allows controlled release of soil bacteria, and protects and stabilizes it, while ensuring longer shelf life. For the practical testing, Cupriavidus necator H16 was chosen as a nitrogen fixing bacteria for the inoculation. Through an ionic gelation method, it was encapsulated into alginate carriers, forming matrix microcapsules. Sodium alginate reacts with the cross-linking agent calcium chloride to form the capsules. The impact on the quality of the product was tested through several variables. What was revealed was that molecular weight of alginate was proven to have a significant impact. Alginate with higher molecular weight was shown to be suitable for the given system. The most desirable environment was distilled water or a TRIS-HCl buffer. However, the TRIS-HCl was unsuitable in cases of retrieving a dry product by lyophilization. Eventually, the viability of encapsulated cells was examined, and it was proven that encapsulated bacteria endure the process of encapsulation in the above-mentioned environment and they were gradually released from the carrier.
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Dissociation behaviour of natural biocolloids
Karbanová, Kateřina ; Sedláček, Petr (referee) ; Klučáková, Martina (advisor)
This diploma thesis is focused on the study of dissociation behaviour of natural biocolloids, namely humic acids and fulvic acids. Humic and fulvic acids are natural, heterogeneous, high molecular weight substances which behave as weakly acidic polyelectrolytes and they have complex not exactly described structure. They are formed by biochemical transformations of organic residues (mainly plants). They are part of the soil, water, peat, sediments and coal. Solubility of humic acids is affected by pH value. The higher the pH value is the higher the solubility is. Fulvic acids are soluble in whole range of pH values. The aim of this diploma thesis is to determine the dissociation constant for the five kinds of humic acids and four kinds of fulvic acids, which have been isolated from various natural sources. These samples were purchased from IHSS. Dissociation constants were determined by the conductometric method and a combination of measurment pH and the content of acidic functional groups in Na2SO4. UV-VIS spectrophotometry method was used to characterize the quality of humic acids and fulvic acids.
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Complex structural characterization of hydrogels based on semi interpenetrating biopolymer networks.
Trudičová, Monika ; Krzyžánek, Vladislav (referee) ; Sedláček, Petr (advisor)
The content of this diploma thesis was testing the applicability of available structural analysis techniques on a appropriate model hydrogel system. The main aim was to evaluate the advantages and disadvantages of selected structural analysis techniques and the influence of the composition change of the hydrogel system on its internal structure. Semi-interpenetrated hydrogels based on agarose of different concentration were chosen as model system, this type of material was chosen for simple and repeatable preparation and also for its application potential. Electron microscopy (SEM, cryoSEM), mercury porosimetry and turbidimetry were chosen as structural analysis techniques. Experimental results could be used to improve knowledge about the influence of hydrogel composition on its structure and the comparison of chosen techniques will be used for the choice of appropriate structural analysis in the future, which will be applicable to other hydrogel systems as well.
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Diffusion characterization of hydrogels at microscopic level
Uher, Tadeáš ; Smilková, Marcela (referee) ; Sedláček, Petr (advisor)
This Bachelor’s thesis deals with study of diffusion in gels on microscopic level. The method of fluorescence correlation spectroscopy is used for the study of diffusion and this method is based on monitoring the diffusion coefficients in a confocal volume of studied substance. As fluorescence probe is used organic pigment called Rhodamin 6G and diffusion experiments are realized in hydrogel matrix, whose medium forms thermoreversible linear polycarbohydrate – agarose, with addition of reactive component – biopolymer called sodium polystyrenesulphate in certain concentrations. The purpose of the thesis is to research the impacts of interaction between diffusing pigment and specific type of hydrogel. The change of final diffusion coefficient is observed. It is assumed, that sodium polystyrenesulphate influences the diffusion of fluorophore (Rhodamin 6G) in agarose hydrogel.
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Diffusion of organic molecules in the hydrogel environment
Holubová, Anna ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with study of hydrogels formed by phase separation of hyaluronan with oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and Septonex. It follows the bachelor thesis and extends the knowledge about the detailed characterisation of the inner environment of the hydrogel by determining the diffusion behaviour of the fluorescent probes Atto 488 and Nile Red using fluorescence correlation spectroscopy (FCS) technique and its modified version dual-focus fluorescence correlation spectroscopy (2f-FCS). Compared results showed that both methods show similar values and probes specifically interact with CTAB but Atto 488 shows only weak interaction with Septonex compared to Nile Red. Additionally, these interactions were not affected by the molecular weight of hyaluronan. In conclusion, it was recommended to measure this type of hydrogel in a small depth of gel using a conventional method.
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Functional technical materials modified by lignin isolated from winery by-products
Chovanec, Martin ; Sedláček, Petr (referee) ; Kovalčík, Adriána (advisor)
In this thesis, the possibility of producing composites of epoxy resin and lignin isolated from grape skins was examined. The motivation was to reduce the amount of bisphenol A, which is nowadays used to produce most of the epoxy resins and is the main environmental pollutant. The composites were prepared by mixing commercial bisphenol A diglycidylether (DGEBA) with lignin samples. In this work, the sodium hydroxide pulping method in a hydrothermal autoclave was used to isolate lignin. The content of reducing sugars, lignin, and fatty acids in the sample of grape skins was determined and the chemical composition was determined by Fourier transform infrared spectroscopy (FT-IR). The chemical and thermal properties of isolated soda lignin were compared with commercial Kraft lignin and analyzed by elemental analysis, FT-IR, thermogravimetric analysis, and ash content determination. Epoxidized lignin was prepared by three methods, which were compared based on yield, epoxide equivalent, and chemical composition. Samples of unmodified soda lignin and epoxidized soda and Kraft lignins were selected for the preparation of epoxy composites. Experiments have shown that isolated soda lignin was not suitable for the addition to epoxy resins, unlike Kraft lignin. Thermal and viscoelastic properties of the prepared composites with a homogeneous structure were analyzed. It was found that the epoxy composites with added epoxidized Kraft lignin showed lower material stiffness and the glass transition temperature was significantly shifted to lower temperatures.
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Corelation of transport and viscoelastic properties of hydrogels with diffusion gradient
Ščotková, Romana ; Sedláček, Petr (referee) ; Smilek, Jiří (advisor)
The aim of the presented bachelor thesis was to prepare hydrogels with diffusion gradient and to demonstrate the gradient structure of hydrogels in terms of viscoelastic and transport properties. With regard to potential biomedical applications, the cationic polysaccharide chitosan was selected for the preparation, especially for its antimicrobial and biocompatible properties. The resulting gradient hydrogels were subsequently characterized by oscillation tests to observe the differences using different concentrations and molecular weights of chitosan, crosslinking agent concentrations, dispersion environments for chitosan dissolution, and different gel crosslinking times. The samples were also subjected to drying experiments to confirm the gradient structure formed. The ability of the hydrogels to transport the active species was confirmed by visually evaluating the permeation of the anionic dye (bromothymol blue) during the gelation process. From the results obtained during the experimental part of the bachelor thesis it can be concluded that the viscoelastic properties of hydrogels can be modified by changing selected parameters during the preparation itself. It was also confirmed the assumption that with better mechanical properties of hydrogels there is a deterioration of transport properties. By controlled diffusion method, it was possible to prepare a hydrogel with a gradual gradient of crosslinking density, thus modifying the viscoelastic and transport properties.
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Structural characterization of hydrogels at macroscopic and microscopic level
Kropáčková, Šárka ; Trudičová, Monika (referee) ; Sedláček, Petr (advisor)
This Bachelor´s thesis deals with study of use several methods for structural analysis of hydrogels. The aim was testing selected method to study structure of these gels. Agarose hydrogels were selected as model systems for their easy preparation and wide use. In this work the oscillatory rheology method was used to study the structure. Strain-sweep and time-sweep tests were performed. These tests were performed in a linear viscoelastic region, in the cross-over area and beyond the cross-over area. It was found that in linear viscoelastic region, the deformation causes the non-covalent bonds and the structure of the gel to break through. Consequently there is relaxation of the polymeric site and increase in elasticity. In cross-over area and beyond cross-over gels does not relax. The gels behave like a fluid.
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Utilization of spectroscopy in study on stress-resistance of bacteria on the sigle-cell level
Köbölová, Klaudia ; Venerová, Tereza (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.
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Utilization of thermal analysis in the study on effects of microbial inhibitors
Bošeľová, Miriam ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the use of thermal analysis in the study on effects of microbial inhibitors. The main aim of this work was to determine the utilization of the method, which is mainly used in different fields of science and research. Three bacterial strains: Cupriavidus necator H16, its mutant strain Cupriavidus necator PHB-4 and Halomonas halophila, were used as model microorganisms. The inhibitory effect of levulinic acid on growth and metabolic activity was monitored by microcalorimetry. It was found that bacteria were able to adapt to levulinic acid to a certain concentration - Cupriavidus necator to 5 g/l and Halomonas halophila to 2 g/l. The thermal analysis results were compared to a conventional method, which is commonly used to study the growth of microorganisms.
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