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Self-encapsulation of Plant Growth Promoting Rhizobacteria as a way towards new generation of bioinoculants
Gašparová, Dominika ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This bachelor thesis focuses on Plant Growth-Promoting Rhizobacteria that can be used as a suitable ecological alternative to conventional fertilizers. However, most of today's approaches to incorporating PGPR into the process of soil fertilization are convoluted and costly. Crosslinking of alginate self-produced by PGPR offers a new possibility of plantgrowth- promoting bacteria encapsulation. The aim of this thesis consists of preparing gel matrices with incorporated bacteria, followed by testing the bacteria's viability and quantity. Firstly, experiments were carried out to determine various properties of gels produced by the chosen representative bacteria Azotobacter vinelandii. These properties include the molecular weight of alginate (with the highest molecular weight of strain CCM 289 (329,78± 6,8) kDa), the alginate's amount in the gel, the content of PHB (34%-44% CDW), and the gel's overall rheological properties. The complex modulus (as a representation of rheological properties) of the firmest gel reached the value of 13,34 kPa. The bacteria content was examined by flow cytometry, the CFU method, and spectrophotometry. The viability of bacteria was determined by analyzing the fluorescence intensity via fluorescent dyes (propidium iodide, fluorescein diacetate, rhodamine 123, calcein AM), then by using the CFU method, and also via the plate method. Furthermore, the effects of various factors such as different pH levels (pH 3, 7, and 10) or adding the specific alginatedegrading enzyme alginase were also examined. Releasing of the bacteria was observed over the course of seven days using flow cytometry and the CFU method. The plate method proved the bacteria's survival after gelation and without any source of carbon. The largest disintegration of gels occurred in acidic pH. Moreover, the effect of alginase on crosslinked bacterial gels and synthetic alginate were very similar. The survival of bacterial cultures in gel and their continuous release are significant findings in the development of novel bioinoculants based on this new concept.
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Microrheology modeling with COMSOL Multiphysics package
Koláček, Jakub ; Sedláček, Petr (referee) ; Pekař, Miloslav (advisor)
This bachelor thesis focuses on modeling Brownian motion using the COMSOL Multiphysics package and its Particle Tracing module. The aim of the work is to design and create elementary models that will be able to suitably simulate the movement of microparticles in viscous and viscoelastic environments, which can later be used for modeling passive microrheology. Within this work, Matlab scripts were created for the calculation of MSD from the simulation results, validation of the viscous model was performed on experimental data and elementary models for the simulation of the viscoelastic environment were also designed. Two different approaches were chosen for the design of these models, namely the use of rigid obstacles under the assumption of a discrete environment and a mathematical model assuming continuous environment. Data from the viscous model showed good agreement with the experimental results. The results of viscoelastic simulations are presented, and further possible development of these models is discussed. The continuous mathematical model is considered closest to modeling viscoelastic behavior because of a characteristic curvature that was observed in the evaluation of MSD.
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Preparation of biodegradable films from PHA copolymers and evaluation of their application potential
Drotárová, Lenka ; Přikryl, Radek (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified.
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Study of hydration of biocolloids
Hývnarová, Lucie ; Sedláček, Petr (referee) ; Klučáková, Martina (advisor)
The subject of this master’s thesis was to examine the hydration properties of selected biocolloids. Specifically, humic substances (fulvic and humic acids), hyaluronic acids in three molecular weights, dextran and chitosan were investigated. Hydration properties of selected biocolloid substances were studied by various methods. The influence of water content during the dissolution of substances and the different time for which water acted on biocolloids were studied. Differential scanning calorimetry DSC measurements resulted in enthalpies and crystallization and melting points. Heat fluxes resulted in the use of perfusion calorimetry. TGA thermogravimetric analysis was used to determine the moisture content of the bioolloids used, after measuring the perfusion calorimeter.
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Utilization of various methods of porosimetry in investigation of internal structure of biopolymer hydrogels
Zahrádka, Jan ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
The main aim of the thesis was to select, optimize and test suitable methods for studying inner structure of hydrogel materials. The study of the size of pores with potential use in fundamental and applied research of these materials is the central focus of the thesis. Firstly, a literature search was done in order to collect basic information about usefulness of the individual methods. On the basis of this literature search, the experimental part of the work was divided into several steps. First, selected methods of conventional porosimetry (mercury intrusion porosimetry, BET analysis and DSC thermoporometry) were applied on reference porous materials and the results obtained by the individual methods were compared. The methods optimized with the use of these reference materials were then utilized in the analysis of porosity of model hydrogels based on polyvinyl alcohol. Based on the results of this analysis, the usability and benefits of the individual methods were discussed with respect to the sample preparation, time and financial demands, and also from the viewpoint of compliance of the determined pore sizes with the range of sizes declared for the physical PVA gels.
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Preparation and characterization of hydrogel delivery systems from poly-gamma-glutamic acid
Mai, Thuy Ha ; Smilek, Jiří (referee) ; Sedláček, Petr (advisor)
The presented diploma thesis focuses on the development and preparation of model poly--glutamic acid based hydrogel carrier systems. For the hydrogel carrier preparation, methods based on physical interactions between polyelectrolyte and oppositely charged multivalent ion or oppositely charged macromolecule were proposed. Based on the pilot experiments, trivalent iron cation and chitosan were selected as suitable crosslinking agents. By crosslinking -PGA via ferric cations a successful bead formation has been accomplished and attempts have been made for a preparation of beads smaller than 1 000 m by employing microencapsulation techniques. Rapid disintegration of -PGA beads occurred upon immersion of the beads in water making these systems unsuitable for drug carrier applications. Interactions between -PGA and chitosan lead to polyelectrolyte complexes formation and by optimizing concentrations and pH of polyelectrolyte solutions, a formation of homogenous looking hydrogel with high water content (>0,9) has been achieved.
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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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Behaviour of microorganisms using microcalorimetry
Piletskaya, Maryia ; Sedláček, Petr (referee) ; Krouská, Jitka (advisor)
The diploma thesis deals with the study of the behavior of a microorganism with the help of microcalorimetry. The theoretical part of the thesis deals mainly with the characterization of the microorganism Rhodospirillum rubrum selected for experimental work and the application of microcalorimetry in microbiology. The second goal was to screen the growth of the bacterial strain R. rubrum using different substrates, and subsequently, based on experimental results, to select the most suitable substrate, and optimize its concentration and aeration of the medium for the microorganism using microcalorimetry. Hexanoate, malate, acetate and fructose were used to examine the most suitable substrate by microcalorimetry. Another goal was data processing and discussion of the advantages and disadvantages of substrate selection and media aeration with respect to bacterial strain growth. Hexanoate was found to be the best substrate for growth, followed by optimization of the concentration and aeration of the medium. The results of the microcalorimetric measurement were confirmed by spectrophotometric study of the growth curve of the investigated bacteria. The effect of osmotic pressure on bacteria and the dependence of their survival on the amount of polyhydroxyalkanoates (PHA) in the cell were also studied. The amount of PHA in the biomass was determined chromatographically.
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Program-controlled freeze drying for the preparation of the delivery system of native hyaluronan and hydrophobic molecules
Waczulíková, Kristína ; Sedláček, Petr (referee) ; Enev, Vojtěch (advisor)
The amphiphilic nature of the hyaluronan biopolymer facilitates interactions between its hydrophobic portions of the chain and the hydrophobic ligands, making it one of the promising compounds that can be used as a suitable carrier for drug delivery. Therefore, the aim of this diploma work was by means of a program-controlled freeze-drying to prepare a system based on native hyaluronan, which would be capable of transporting hydrophobic drugs. Perylene, pyrene, prodan and 1,6-diphenyl-1,3,5-hexatriene fluorescence probes were used as model compounds to simulate hydrophobic drug-hyaluronan interactions. Freeze-drying efficiency was studied using infrared spectrometry and scanning electron microscopy. ATR-FTIR spectrometry confirmed for all probes but pyrene the hydrophobic interactions between the hyaluronan and probe molecules based on the presence of the C=C bond valence vibration absorption band in the aromatic probe rings. From the IR spectra, it was not possible to confirm with certainty the presence of residual tert-butyl alcohol in the samples. SEM photographs revealed that increasing the pressure during drying process had a positive effect on the quality of the lyophilized sample in the second series of samples. Interactions of individual fluorescent probes with hyaluronan were studied using steady-state and time-resolved fluorescence spectrometry. Positive results have been obtained, indicating the presence of fluorescent probes interacting with the hydrophobic regions of the hyaluronan chains. The results of this work can contribute to the development of carrier systems in the field of targeted drug distribution.
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