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The utilization of atomic force miscroscopy to study of selected bacterial strain
Nováková, Simona ; Slaninová, Eva (referee) ; Smilek, Jiří (advisor)
The subject of the bachelor’s thesis was the study of selected bacterial strains by a selected method of atomic force microscopy. Thermophilic purple bacteria – Rhodoblastus acidophilus, Rhodobaca barguzinensis – were investigated. The main aim of this theses was focused on optimizing the usage of this microscopic method for imaging in an aqueous environment with the most appropriate fixation of sample. Focusing on bacterial strains demanded to preserve the viability of microorganisms and to observe optimal conditions for maintaining the native state during the measurement process. Furthermore, mechanical properties were evaluated – Young’s modulus and adhesion. The morphology of individual samples was compared for samples of the same species in different pH liquid environments and in environments with different concentrations of dissolved NaCl salt. The ability to produce PHA granules in purple bacteria was also targeted, however, due to the absence of first-class PHA synthase, PHA was not detected. The effects of different cultivation parameters such as different cultivation time or different pH of the environment were compared. Imaging of bacteria was carried out using the SNL-10 tip, which is suitable for bacterial samples in a liquid environment, and the measurement itself was carried out in QITM mode. A glass substance covered with a layer of Poly-L-lysin was used for immobilization. Finally, the mechanical properties were evaluated through Young’s modulus and adhesion for samples with various environmental factors.
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Biosynthesis of polyhyroxyalkanoates in non-suflur purple bacteria
Fiala, Tomáš ; Vodička, Juraj (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis focuses on polyhydroxyalkanoates (PHA) and their production using non-sulfur purple bacteria, specifically Rhodospirillum rubrum. The cultivation conditions were optimized especially with respect to the determination of NaCl concentrations for the following evolution experiments. Then this microorganism was repeatedly cultured at a high concentration of NaCl (40 g/L). Passage system was used in this cultivation with 48 h between inoculations under aerobic conditions in the dark. In the samples cultivated in this way, the growth rate was investigated using optical density and the amount of biomass and PHA using gas chromatography with an FID detector. As the last experiment PCR analysis was performed to test the 16S rRNA and phaC genes. The conclusion of the work is the successful adaptation of R. rubrum to a concentration of 40 g/l NaCl. Furthermore, the positive effect of stress stimulus on the increased PHA production was proven.
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Optimization of the cultivation media for thermophilic bacterium Caldimonas thermodepolymerans
Fajtová, Zuzana ; Černayová, Diana (referee) ; Obruča, Stanislav (advisor)
The subject of the bachelor ‘s thesis is the optimization of the cultivation conditions of the thermophilic bacterium Caldimonas thermodepolymerans. The theoretical part provides a general characterisation of PHA materials, their synthesis, degradation, and applications. In addition, extremophilic microorganisms and the bacterium C. thermodepolymerans are discussed in the theoretical part. The experimental part of the thesis focuses on the production of the PHB by the bacterium C. thermodepolymerans DSM 15344. A series of experiments were carried out to optimise the cultivation conditions. The influence of temperature, inoculum ratio, inoculum age, mineral medium composition, and concentrations of nitrogen sources in the mineral medium on culture growth and PHB production was investigated. It was found that the original conditions set were already close to optimal for the cultivation. A higher yield compared to the control conditions was observed with a combination of 0.5 g/l yeast extract and 1.5 g/l ammonium chloride, resulting in a 7% increase in PHB.
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Prediction of small RNAs in selected bacterial producers of bioplastics
Heřmánková, Kristýna ; Vítková, Helena (referee) ; Sedlář, Karel (advisor)
Využití bakterií v biotechnologii vyžaduje důkladné pochopení schopností použitých bakterií pro maximalizaci výtěžku. To platí například pro výrobu bioplastů, kde konkurenceschopnost s levnějšími plasty na bázi fosilních paliv závisí také na ceně procesu pro získání požadovaného výtěžku. Nahlédnutí do regulačních mechanismů bakterií, jako je regulace pomocí malých RNA, která zajišťuje rychlou reakci především na stresové podmínky, může pomoct odhalit jejich vlastnosti a posoudit tak jejich využitelnost v biotechnologiích. Tato diplomová práce se zabývá zpracováním bakteriálních transkriptomických dat, které byly sekvenovány pomocí RNA-Seq. Práce je zaměřena zejména na predikci malých RNA u poskytnuté bakterie, slibného kandidáta pro využití v biotechnologii. Cílem je získat přehled o malých RNA v této bakterii, zejména analyzovat potenciální regulaci syntézy PHA malými RNA, které by mohly rozšířit přehled o využitelnosti bakterie pro produkci PHA.
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Biocrystallization as a new concept in development of piezoelectronics for use in biomaterials
Dobešová, Kateřina ; Plichta, Tomáš (referee) ; Sedláček, Petr (advisor)
This thesis focuses on biofilament methods and biocrystallization of bacterial cells producing polyhydroxyalkanoates, specifically the bacterium Cupriavidus necator H16. The main techniques used for cell orientation include spin-coating and acoustic waves. Biocrystallization was induced by temperature, osmotic, and acid stress. The degree of cell orientation was monitored by an atomic force microscope, and crystallization was determined by FTIR. Among other parameters, the thickness of the layers was evaluated using a profilometer. The amount of PHA in the bacterial cells was determined by gas chromatography. The preparation of crystalline ordered layers of biomaterials represents a route to piezoelectric biomaterials.
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Involvement of vibrational spectroscopy in the in-situ analysis of PHA in bacterial biomass.
Kevélyová, Barbora ; Samek, Ota (referee) ; Sedláček, Petr (advisor)
The aim of this thesis was the study of polyhydroxyalkanoates in bacterial biomass using the ATR-FTIR method. Furthermore, the PCA method was chosen to evaluate the measured data and observe correlations or differences among the spectra of various biomass samples. The bacterial producents were Aneurinibacillus thermoaerophilus H1, AH30 and AFn2, Cupriavidus malaysiensis DSM 25816, DSM 19416 and DSM 19379 and three strains of Schlegelella thermodepolymerans M 15344, DSM 15264 and LMG 21645. Various heteropolymers with different monomer content and total PHA content were investigated. The monomers in the samples were 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxyvalerate, 5-hydroxyvalerate and 4-hydroxyhexanoate. As the reference method for accurate quantitative and qualitative determination of polyhydroxyalkanoates in the samples, gas chromatography was performed. The spectra obtained by the FTIR method were compared and significant peaks related to the qualitative properties of PHA, namely monomer composition and crystallinity, were searched for. The PCA statistical method was successfully applied to the collected data from FTIR, with the help of which the qualitative differences between the samples were monitored. The most significant differences were observed in the presence of monomers 4-hydroxybutyrate and 4-hydroxyhexanoate, and they were related to the crystallinity of the samples. The analysis did not show potential for semi-quantitative determination of PHA in biomass. Differences between microorganisms were not observed. The procedure for evaluating FTIR spectra using PCA could in the future be used in the study of biomass containing various monomeric units, especially hydroxyvalerates in case of their higher content within the sample.
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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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The influence of electrochemically generated hydrogen peroxide on the stress response of microorganisms for the purpose of increased PHA production
Najbrtová, Johana ; Nováčková, Ivana (referee) ; Ehlich, Jiří (advisor)
The subject of the presented thesis is the effect of electrochemically formed hydrogen peroxide on the production of polyhydroxyalkanoate. In this work, hydrogen peroxide was generated directly in the medium with bacteria by two-electron reduction of oxygen. This thesis is divided into a theoretical part, which is devoted to approaching the topic of polyhydroxyalkanoate, oxidative stress, electrochemical production of H2O2 and the connection of electrochemistry with biotechnology. The experimental part is focused on the application of these findings. Hydrogen peroxide was generated by applying an electrical potential to an electrode system composed of a stainless-steel working electrode, a titanium counter electrode, and a pseudo-reference Ag/AgCl electrode embedded in a polypropylene holder. After initial electrochemical characterization of the prepared system, a potential of 0,8 V and -1 V was selected for application in cultivation experiments. Here, the effect of H2O2 on the stress response of the bacterium Cupriavidus necator H16 from the Czech collection of microorganisms was investigated. From the results of the cultivation experiments, it was evident that the electrochemically produced H2O2 does not have a significant effect on the development of the bacterial culture. However, the electrode system itself positively affects bacterial growth. The influence of H2O2 was not noticeable due to the low production in medium. This was caused by a low concentration of oxygen in the medium. During this work, a new electrode system with higher H2O2 production was designed, which could use atmospheric oxygen for reduction to H2O2. However, even with this system, a desired stress response was not achieved, because increased production of H2O2 was prevented by the formation of biofilm on the electrodes. Furthermore, it was found that the materials used for the electrode system support the formation of biofilm, mainly titanium, stainless-steel mesh and polypropylene.
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Production of polyesters by extremophilic bacteria
Bednárová, Kristína ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of the production of polyhydroxyalkanoates using thermophilic bacterial strains Schlegelella thermodepolymerans, Schlegelella aquatica and Thermomonas hydrothermalis. The aim of the thesis is to optimize the conditions of polymer production. In bacterial strains of the genus Schlegelella, phaC gene at the genotype level was confirmed. The ability to produce polyhydroxyalkanoates in the bacterial strain Thermomonas hydrothermalis at the phenotype level was demonstrated. The utilization of selected carbon sources was studied experimentaly. The production of PHA was optimized by selecting the appropriate substrate and cultivation temperature. The effect of precursors on the production of copolymers P(3HB-co-4HB) and P(3HB-co-3HV) was observed. The most promising carbon substrate for bacterial strain Schlegelella aquatica was glycerol while the best metabolic activity was at 45 °C. Strain Thermomonas hydrothermalis was able to produce the copolymer P(3HB-co-4HB) with extreme high molar fraction of 4HB. Optimal cultivation temperature was 55 °C. The highest yield of P(3HB) among selected bacteria, synthesized Schlegelella thermodepolymerans cultivated on xylose at 55 °C. For this strain, the effect of aeration on biomass growth and PHA production was observed. In an effort to reduce production costs, Schlegelella thermodepolymerans was cultivated on model hydrolysates of lignocellulose-rich materials. Finally, batch cultivation in a larger production volume in laboratory fermentor was performed.
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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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