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Involvement of polyhydroxyalkanoates in stress response of bacteria during late stationary phase
Šuráňová, Zuzana ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase. Bacteria Cupriavidus necator H16 (able to produce PHA) and bacteria Cupriavidus necator H16/PHB-4 (unable to produce PHA) were used for the experiment. In the theoretical part, the polyhydroxyalkanoates and a stress response of bacteria were reviewed. In the experimental part of the work, the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase against selected stress factors was studied. A resistence against various stress conditions of bacteria was studied. During long term cultivations a culture viability as well as PHA distribution among bacterial populations were assessed by using flow cytometry and the PHA content in biomass was analyzed by gas chromatography with FID detector.. Based on the results obtained in this work, it was found that the PHA acumulating bacteria Cupriavidus necator H16 is capable to survive carbon substrate limitations better than the bacteria Cupriavidus necator H16/PHB. Further, Cupriavidus necator H16 also revealed higher resistence against various stress factors such as ethanol treatment and freezing.
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Involvement of polyhydroxyalkanoates into stress response of bacteria
Kučera, Dan ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates (PHA) into stress response of bacteria. The theoretical part of the thesis deals with the possibility of establishing the viability of microorganisms using modern techniques, in particular flow cytometry. Furthermore, the research focused on selected stress factors and PHA involvement in stress response was described. In the experimental part of the work the stress response with regard to the ability to accumulate PHA was assessed. Bacteria Cupravidus necator was used for the experiment. Its ability to accumulate PHA at a later stage of growth increased resistance to ethanol, high temperature and freezing. Conversely, the PHA-producing strain showed lower resistance to the action of inorganic acids and bases. This may be caused by different morphology of PHA-producing cells. One of partial objectives was also to study the possibilities of staining of living cells accumulating PHA using Nile red. The research proved that the dye penetrates into living cells at elevated temperature of 40-45°C. This temperature is not lethal to the cells and the intensity of staining is sufficient to distinguish PHA-producing cells using flow cytometry; that can be applied in the selection of industrial PHA producers.
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Connection between stress resistance of bacteria to temperature-mediated stress and PHA accumulating ability
Nováčková, Ivana ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the involvement of polyhydroxyalkanoates (PHA) into the stress response of bacteria, specifically when they are exposed to temperature-mediated stress. The theoretical part focuses on the stress response of bacteria in general, the mechanism of response to temperature-mediated stress and PHA involvement into the stress response. The aim of experimental work was to study the correlation between the amount of PHA in the biomass of the bacteria and their viability after exposure to temperature-mediated stress. PHA producing bacterial strains Cupriavidus necator H16, Burkholderia cepacia and Burkholderia sacchari and non-producing mutant strain Cupriavidus necator PHB4 were used for experimental work. Bacteria accumulated poly(3-hydroxybutyrate) (PHB), the amount of PHB in the biomass was determined by gas chromatography with FID. Viability of live bacterial cells and cells after exposure to temperature-mediated stress was determined by using flow cytometry. It turned out that the content of PHB in biomass assists bacteria to resist the freezing shock. By comparing the viability of bacteria C. necator H16 and C. necator PHB4 after exposure to multiple stresses it also has been observed that PHB increased the resistance of cells against temperature-mediated stress and stress caused by low pH.
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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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Metabolic and biophysical characterization of bacterial cells capable of PHA accumulation
Slaninová, Eva ; Lehocký, Marián (referee) ; Doškař, Jiří (referee) ; Chodak, Ivan (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.
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Electrochemical evolution of hydrogen peroxide for biotechnological applications
Runštuková, Nikola ; Kratochvíl, Matouš (referee) ; Ehlich, Jiří (advisor)
This master‘s thesis deals with the connection of two distinct fields, electrochemistry and biotechnology, where electrochemically generated peroxide was subsequently used in bacterial cultivation. The peroxide was synthesized in an electrolytic cell by reducing oxygen at the cathode to which suitable electrical potential was applied. The aim of this thesis was to investigate the dependence of the amount of hydrogen peroxide produced on a few selected parameters, such as cathode potential, electrode material, the geometry of the measuring cell, or the stirring speed, and then to apply these findings in the continuous stressing of the bacterium Cupriavidus necator H16, in order to increase the production of polyhydroxyalkanoates. The selected parameters were first tested in small-scale measuring cells and then experiments were carried out on a larger scale, in Erlenmeyer flasks. During the experiments, the amount of electric current produced was measured and also the peroxide concentration was examined spectrophotometrically along with the oxygen concentration measured by using an optical sensor. Based on the obtained results, bacteria were cultivated in flasks with a built-in system of steel electrodes, due to which oxidative stress in the form of in situ generated hydrogen peroxide was continuously applied. The bacterial cultures were evaluated by measuring the change in optical density, gravimetric analysis and GC-FID analysis. It was found that even small amounts of hydrogen peroxide (~25 M) can induce a stress response in the organism in the form of increased polyhydroxybutyrate (PHB) synthesis. A percentage increase in the PHB content of the biomass was achieved, however, the overall polymer yield was lower due to lower biomass growth.
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Metabolic and biophysical characterization of bacterial cells capable of PHA accumulation
Slaninová, Eva ; Lehocký, Marián (referee) ; Doškař, Jiří (referee) ; Chodak, Ivan (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.
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Connection between stress resistance of bacteria to temperature-mediated stress and PHA accumulating ability
Nováčková, Ivana ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the involvement of polyhydroxyalkanoates (PHA) into the stress response of bacteria, specifically when they are exposed to temperature-mediated stress. The theoretical part focuses on the stress response of bacteria in general, the mechanism of response to temperature-mediated stress and PHA involvement into the stress response. The aim of experimental work was to study the correlation between the amount of PHA in the biomass of the bacteria and their viability after exposure to temperature-mediated stress. PHA producing bacterial strains Cupriavidus necator H16, Burkholderia cepacia and Burkholderia sacchari and non-producing mutant strain Cupriavidus necator PHB4 were used for experimental work. Bacteria accumulated poly(3-hydroxybutyrate) (PHB), the amount of PHB in the biomass was determined by gas chromatography with FID. Viability of live bacterial cells and cells after exposure to temperature-mediated stress was determined by using flow cytometry. It turned out that the content of PHB in biomass assists bacteria to resist the freezing shock. By comparing the viability of bacteria C. necator H16 and C. necator PHB4 after exposure to multiple stresses it also has been observed that PHB increased the resistance of cells against temperature-mediated stress and stress caused by low pH.
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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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Involvement of polyhydroxyalkanoates in stress response of bacteria during late stationary phase
Šuráňová, Zuzana ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase. Bacteria Cupriavidus necator H16 (able to produce PHA) and bacteria Cupriavidus necator H16/PHB-4 (unable to produce PHA) were used for the experiment. In the theoretical part, the polyhydroxyalkanoates and a stress response of bacteria were reviewed. In the experimental part of the work, the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase against selected stress factors was studied. A resistence against various stress conditions of bacteria was studied. During long term cultivations a culture viability as well as PHA distribution among bacterial populations were assessed by using flow cytometry and the PHA content in biomass was analyzed by gas chromatography with FID detector.. Based on the results obtained in this work, it was found that the PHA acumulating bacteria Cupriavidus necator H16 is capable to survive carbon substrate limitations better than the bacteria Cupriavidus necator H16/PHB. Further, Cupriavidus necator H16 also revealed higher resistence against various stress factors such as ethanol treatment and freezing.
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