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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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Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
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Utilization of vibrational spectroscopy for in-situ qualitative and quantitative analysis of polyhydroxyalkanoates in microbial biomass
Kevélyová, Barbora ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis was to evaluate the applicability of selected methods of vibrational spectroscopy for qualitative and quantitative analysis of biomass, which contains polyhydroxyalkanoates. Fourier-transform infrared spectroscopy, that uses attenuated total reflection technique (ATR-FTIR), was chosen for this experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) has been studied in Cupriavidus malaysiensis, Thermomonas hydrothermalis and Aneurinibacillus thermoaerophilus sp. H1. Data were obtained by evaluating the infrared spectra, which were then compared with the results of performed gas chromatography. Visual changes in the spectra were also observed along with crystallinity. No correlation was detected between the data obtained by gas chromatography and by the method ATR-FTIR in quantitative analysis. Thus, this method is not suitable for determining the content of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in biomass in the set experiment and the bacteria used. On the other hand, the method was successfully used for in-situ qualitative analysis of copolymer in the samples of Aneurinibacillus thermoaerophilus sp. H1. The degree of data correlation was relatively high in the given experiment. With these findings, ATR-FTIR is a suitable analytical method for determining the monomer composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in this strain. Comparison with the PHA producer Cupriavidus malaysiensis showed that this method is not universal for all types of microorganisms observed in this thesis.
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Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
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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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Preparation and application particles from copolymer of P(3HB-co-4HB)
Ostanina, Kseniia ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with preparation of particles from poly(3-hydroxybutyráte-co-4-hydroxybutyráte). Copolymer was dissolved in acetone and then the solution had been dosed into the anti-solvents. Acetone and anti-solvent are perfectly miscible. Theoretical part focuses on the properties of the copolymer, its production and potential use in medical applications. In the experimental part the copolymer was dripped into selected anti-solvents by syringe and pipette above and below the surface of anti-solvent. The following anti-solvent were chosen: water, methanol, dimethylsulfoxid (DMSO), 50 % acetone-water, 75% acetone-water, 50 % methanol-water, 75 % methanol-water mixtures. There were three copolymers used with different compositions of 4HB units (63 %, 87 % a 33 %). Copolymer composition was analyzed by gas chromatography. Copolymers were prepared by bacterial strain Aneurinibacillus sp. H1. Due to the results particles of size approximately 100-500 nm can be prepared by methods described in this work. The produced particles can be successfully used in medical applications.
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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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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
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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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Utilization of physicochemical and spectroscopic techniques in study of stress-response of cyanobacteria
Skoryk, Maksym ; Šedrlová, Zuzana (referee) ; Sedláček, Petr (advisor)
Tato bakalářská práce je soustředěná na zkoumaní cyanobakterií, vystavených hyper a hyposmotickým stresům. Na základě literární rešerše byly navžené vhodné analytické metody určené ke studiu dvou modelových organismu - Synechocystis sp. PCC 6803 a Synechocystis sp. salina Wislouch CCALA 192. Celkem čtyři metody byly použité k charakterizaci osmoticky zatížených bakterií. Průtoková cytometrie byla použita k vyhodnocení viability. Fluorescenční sonda SYTOX Blue poskytla důvěryhodnou informaci ohledně viability bakterií. Propidium jodid naopak poskytl nezřetelnou informaci. Optické vlastnosti cyanobakterií byly prozkoumané pomoci UV-VIS absorpčních a turbidimetrických měření. Termogravimetrická analýza byla použita pro mapování změn obsahu vody v osmoticky stresovaných buňkách. Tato metoda ukazala. že PHB-positivní Synechocystis sp. PCC 6803 jsou pravděpodobně vice odolné vůči hyperosmotickým stresům než PHB-negativní. Plynová chromatografie byla použita ke kvantifikaci vnitrobuněčného PHB, který činil přibližně 1-2 % suché hmoty PHB-positivních buněk.
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