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Utilzation of physico-chemical analyses in study on microbial stress-response
Slaninová, Eva ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the utilization of physico-chemical analysis in study on microbial stress–response. The main content of this work was to propose and optimize techniques and methods which are generally used in different industries. Two bacterial strains, Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4 producing polyhydroxyalkanoates (PHA) under certain conditions, were used as model microorganisms. Initially, microscopic teques such as transmission electron microscopy (TEM), cryo scanning electron microscopy (cryo SEM) and atomic force microscopy (AFM) were proposed and tested for the characteriozation of morphological differences of bacteria. Furthermore, viscoelastic properties of bacteria were determined and compared by the oscillatory tests of the rheology method considering another type of samples. Thermal analysis methods, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in particular, focused on water transport and behavior of intracellular water influenced by presence of the granules of PHB in bacteria. The last technique, utilized in the study, was dynamic and electrophoretic light dispersion during pH changes for characterization of the surface properties of bacteria such as size, zeta potential and the isoelectric point.
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Impact of PHA accumulation on stress resistance of bacteria against disinfection agents
Krupičková, Kristýna ; Fryšová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the influence of acumulation of PHA balancing on the resistence of bacteria against desinfecting means. Two bacterial strains, PHA accumulation Cupriavidus necator H16 and its mutant strain insoluble PHA synthase Cupriavidus necator PHB-4 were used as model microorganisms. These bacteria were exposed to three different concentration of three desinfecting means (etanol, savo and ajatin). The viability of bacteria by the action of disinfectants was determined by the plate method and by the flow cytometry. The influence of desinfecting means against bacteria was examined by the broth dilution method which rested in measurement of absorbance over time. Agar diffusion method was the last one technique use. The results show that PHA production positively aids cell defence against desinfectants because bacterial strain Cupriavidus necator H16 is more resistant than its mutant strain Cupriavidus necator PHB-4. Of the free selected disinfectants, ajatin is the most effective.
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Stress resistance of PHA accumulating bacteria against conditions associated with gastrointestinal tract
Kovářová, Radka ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.
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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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Crystallization of microbial polyesters - methods and biological consequences
Liczka, Jan ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis was to test suitable methods for preparation of amorphous form of microbial polyesters and to perform model experiment to study a kinetics of crystallization of prepared polyesters. The theoretical part deals with PHA characteristics and various methods of isolating PHB granules from bacterial cells. In the experimental part, measuring techniques such as analytical centrifugation, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were tested to characterize the properties of PHB granules. Furthermore, an experiment was performed to isolate native PHB granules from Cupriavidus necator using lysozyme, deoxyribonuclease and cell disruption by ultrasonification. The results of the analyzes showed that the granules were either isolated amorphous only in the wet and crystallized upon drying, or that the granules were completely amorphous but not isolated from the bacterial cell.
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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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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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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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Intrinsic fluorescence of bacteria Cupriavidus necator
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.
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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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