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Characterization of surface and mechanical properties of microbial cells
Khýrová, Markéta ; Lukeš,, Jaroslav (referee) ; Plichta, Tomáš (advisor)
The subject of this diploma thesis is the characterization of bacterial strains capable of polyhydroxyalkanoates (PHA) production in terms of morphology and mechanical properties. With this aim, an atomic force microscope (AFM) was used together with a nanoindentation instrument to perform microcompression tests on selected microorganisms. The model bacterial strains, Cupriavidus necator and Rhodospirillum rubrum, were tested in their PHA-producing form and in their mutant non-producing variant, in order to study the effect of PHA amount on bacterial morphology and mechanical properties. In the experimental part of this work, the preparation of native bacterial samples for both techniques were first optimised. Glass substrates were treated with plasma and the cells were subsequently fixed with poly-L-lysine. In addition, sample preparation including fluorescent staining was optimized for the microcompression tests in order to easily detect cells and determine their size. Finally, the impact of different measurement parameters and mathematical models for calculating Young's modulus on the AFM data were evaluated. The results revealed the effect of PHA on bacterial cell morphology and showed that PHA producers exhibit higher Young´s modulus on average compared to their mutant. It was also shown experimentally that microcompression tests provide significantly higher Young's modulus values than those obtained by AFM.
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Study of biological samples using atomic force microscopy
Khýrová, Markéta ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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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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Study on metabolism of polyhydroxyalkanoates in Rhodospirillum rubrum
Rubanová, Blanka ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoate (PHA) metabolism in Rhodospirillum rubrum under different cultivation conditions. The conditions differed in the aeration rate (aerobic, anaerobic, microaerobic) and in the presence of a light source (light cultivation, dark cultivation). First, different volumes of medium were suggested and tested for the fastest and the most suitable inoculum cultivation. Subsequently, cultures of Rhodospirillum rubrum under different conditions in terms of PHA content were compared by gas chromatography. In addition to gas chromatography, other complementary methods were used for better characterization of bacterial growth and metabolism, such as flow cytometry, UV-VIS spectroscopy (transmission and diffusion transmission modes). Fluorescence microscopy and cryo-scanning electron microscopy were used in terms of cell morphology. Furthermore, the ability of bacteria to synthesize color pigments (carotenoids and bacteriochlorophyll a) was investigated. The bacteria were cultivated on different types of media (liquid, solid), the color pigments were subsequently extracted, and their concentrations were calculated. UV-VIS spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) were also used to measure color pigments.
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Characterization of morphological and physico-chemical properties of microorganisms and components of cell by atomic force microscopy
Blaňka, Ondřej ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The aim of this bachelor thesis was to characterize Rhodospirillum rubrum bacterial cells that produce polyhydroxyalkanotate (PHA) and Rhodospirillum rubrum phaC without the ability to produce intracellular PHA granules using atomic force microscopy. Furthermore, isolated polyhydroxyalkanoate granules in their native state were also characterized by this method. Poly-L-lysine was used in the preparation of the samples, specifically for fixation of bacterial cells and isolated PHA granules, and was applied to slides that had been exposed to plasma at a concentration of 0.01 mg/ml. The tip tested was the MLCT - A tip. The first characteristics obtained were topographic images of Rhodospirillum rubrum and Rhodospirillum rubrum phaC cells and isolated PHA granules. The bacterial cells and PHA granules were further characterized using Young's modulus. To obtain it, a method was designed in which whole cells and PHA granules were first imaged, respectively, and then, in the case of cells, measurements were made targeting individual locations on the cell. The measured data were first evaluated in JPK Data Processing software using the Hertzian data approximation model. This evaluation proved to be unreliable, so a second method was devised using the directive lines obtained with the steep parts of the "extend" curve to evaluate. Whole-cell measurement was chosen as a more reliable measurement because it yielded smaller deviations and provided information about the entire surface of the cell or granule. The Rhodospirillum rubrum directive was (4.5±1.5) nN/µm and for Rhodospirillum rubrum phaC was (14.1±4.4) nN/µm. This confirmed the hypothesis that Rhodospirillum rubrum cells containing PHA granules exhibit different stiffness compared to Rhodospirillum rubrum phaC cells without the presence of PHA granules. The directive for isolated PHA granules was determined to be (9.0±1.0) nN/µm. When compared with the directives of bacterial cells, it shows that the PHA granules exhibit a stiffness that lies between that of Rhodospirillum rubrum and Rhodospirillum rubrum phaC.
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In-situ analysis of photosynthetic pigments in bacterial cells
Janošec, Juraj ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor's thesis deals with the design and characterization of an appropriate combination of methods for the analysis of photosynthetic pigments in a model organism that produces such pigments. Specifically, the microorganism Rhodospirillum rubrum was investigated, which, in addition to photosynthetic pigments, also produces significant products such as potential plastic substitutes called polyhydroxyalkanoates. Structural and quantitative composition of the pigments was analyzed under different cultivation conditions with respect to light conditions. The methods used included spectroscopic techniques, particularly UV-VIS spectroscopy. Specifically, single-beam and double-beam UV-VIS spectroscopy in transmission and diffuse transmission modes, as well as fluorescence spectroscopy, were employed. These methods confirmed the presence of bacteriochlorophyll and several carotenoids, with the highest representation being of spirilloxanthin.
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Study on metabolism of polyhydroxyalkanoates in bacterium Rhodospirillum rubrum
Pacasová, Viktorie-Alexandra ; Byrtusová, Dana (referee) ; Slaninová, Eva (advisor)
The present thesis focuses on polyhydroxyalkanoate (PHA) production in Rhodospirillum rubrum, comparing wild-type PHA-producing (R. rubrum) and mutant PHA-non-producing strains in terms of cell morphology. These bacterial strains were also subjected to selected stresses and the effect of PHA on the stress response was determined using advanced analytical techniques. The two strains used were first cultured under different conditions differing in light access, oxygen, culture time and substrate. The effect of culture conditions and PHA content on resistance to selected stressors - elevated temperature, osmotic stress and repeated freezing cycles - was then investigated. First, strains were cultured under aerobic conditions in the dark for 48 and 72 h. Viability of these samples was examined using a fluorescent probe (propidium iodide) by flow cytometry, while cell disruption was examined by electron microscopy. Furthermore, a similar experiment was performed in which the wild-type and mutant strains were cultured under microaerobic conditions in the light for 120 h to produce pigments (bacteriochlorophyll a and carotenoids). UV-VIS spectroscopy was used to determine the extracted pigments. The effect of culture time and conditions on the stress response was also determined for both bacterial strains by flow cytometry and electron microscopy. In the experimental part of the work, the ability to produce the copolymer P(3HB-co-3HV) was confirmed in a wild-type strain of R. rubrum. The hypothesis of the protective property of PHA granules when exposed to freezing cycles was also confirmed. In contrast, the protective function of PHA was not proven when exposed to osmotic stress.
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Study of biological samples using atomic force microscopy
Khýrová, Markéta ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.
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Study on metabolism of polyhydroxyalkanoates in Rhodospirillum rubrum
Rubanová, Blanka ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoate (PHA) metabolism in Rhodospirillum rubrum under different cultivation conditions. The conditions differed in the aeration rate (aerobic, anaerobic, microaerobic) and in the presence of a light source (light cultivation, dark cultivation). First, different volumes of medium were suggested and tested for the fastest and the most suitable inoculum cultivation. Subsequently, cultures of Rhodospirillum rubrum under different conditions in terms of PHA content were compared by gas chromatography. In addition to gas chromatography, other complementary methods were used for better characterization of bacterial growth and metabolism, such as flow cytometry, UV-VIS spectroscopy (transmission and diffusion transmission modes). Fluorescence microscopy and cryo-scanning electron microscopy were used in terms of cell morphology. Furthermore, the ability of bacteria to synthesize color pigments (carotenoids and bacteriochlorophyll a) was investigated. The bacteria were cultivated on different types of media (liquid, solid), the color pigments were subsequently extracted, and their concentrations were calculated. UV-VIS spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) were also used to measure color pigments.
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