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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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Growth screening of Rhodospirillum rubrum using microcalorimetry
Vetchý, Antonín ; Slaninová, Eva (referee) ; Krouská, Jitka (advisor)
The presented work deals with the study of the growth of the bacterium Rhodospirillum rubrum using microcalorimetry. The theoretical part is devoted to the characterization of the investigated microorganism and the description of the main measurement method. Two strains of the mentioned bacterium were selected for the measurement – the wild type strain and the mutant strain with a deletion of the gene for PHA synthase. Firstly, the optimization of measurement conditions was performed, including the examination of various values of the initial optical density. The main objective of the work was to compare the growth of both strains of R. rubrum on different substrates differing in carbon source. Normalized heat flow curves obtained by microcalorimetry, and growth curves measured by the traditional spectrophotometric method were used for comparison. In conclusion, replicable measurement conditions were established, providing comparable results for both strains of bacteria. The most suitable medium for biomass growth of both strains of the bacterium Rhodospirillum rubrum appears to be the SYN FAY medium, while the least suitable medium seems to be the SYN AY medium containing acetate, where partial inhibitory effects on bacterial growth were observed. The results of the analysis using the microcalorimeter were further confirmed and extended by measuring growth curves on an ELISA reader.
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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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Use of molecular techniques for study of thermophilles
Dvořáková, Dominika ; Kouřilová, Xenie (referee) ; Buchtíková, Iva (advisor)
Polyhydroxyalkanoates are microbial storage polymers that represent a green alternative to petrochemical plastics. However, their high production cost limits wider industrial utilization. Use of waste materials as a substrate and/or utilization of extremophilic organisms is an option to reduce production costs. The strain of thermophilic bacteria Aneurinibacillus appears to be a promising producer because of its ability to synthesize wide range of non traditional copolymers. In addition, PHA synthesis is independent of nutrient limitation, which was the subject of this study. Experiments were focused on the expression of genes involved in PHA synthesis in the bacterium Aneurinibacillus sp. AFn2 on different types of production media. The presence of selected genes was verified using classical PCR. After that the cultivation experiments were performed on mineral and complex media to simulate environments with different nutrient availability. Biomass concentration was determined gravimetrically from the samples collected in time during the growth curve measurement. PHA content was measured simultaneously using gas chromatography. Finally, the expression of the studied genes was analysed by RTqPCR. Significant differences were observed in the transcription of individual genes depending on the media type and time. The results confirmed the ability of Aneurinibacillussp.AFn2 to produce PHA independently on nutrient limitation, however, the production rate and cell filling depended on the media type. This fact may be related to the different course of expressed genes involved in PHA synthesis. A higher percentual yield of P(3HB) in cells were observed in complex media along with an increasing trend in the overall gene expression. On the other hand, in mineral medium, the transcription of individual genes only decreased after initial strong expression, and the cell filling of P(3HB) was also lower. The findings lead to understanding not only the expression of selected genes but also the overall synthesis of PHA. At the same time, it offers the way to optimize production with the aim of reducing cost and expanding their market applications.
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Biotechnological production of polyhydroxyalkanoates by thermophiles
Kouřilová, Xenie ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHAs) are microbial biopolymers that may provide a partial alternative to petrochemically produced plastics. Their main advantages are biodegradability, biocompatibility, and the possibility of production from renewable sources. However, the cost of their production is still higher than that of petroleum-based polymers. To increase the competitiveness of biotechnological processes, the concept of Next Generation Industrial Biotechnology (NGIB) has been introduced by other authors. This concept relies on the use of extremophilic microorganisms. When extremophiles are implemented in fermentation processes, the robustness of these technologies is increased and at the same time sterility requirements can be reduced. In the context of PHA production using extremophiles, a subset of halophilic microorganisms is relatively well mapped. Significantly less information is available on another interesting category, namely thermophiles. In line with the idea of NGIB, it is the production of PHAs by thermophilic bacteria that is the focus of this thesis. Attention is paid mainly to the genera Caldimonas, Rubrobacter, and Tepidimonas. For several tested representatives of these genera, the parameters investigated were the optimal cultivation temperature, suitable carbon substrate and the ability to produce copolymers. Based on this basic screening parameters, the most promising producers were selected and subjected to further experiments. Representatives of the genus Rubrobacter have the advantage of being gram-positive non-sporulating bacteria, as the risk of contamination of the isolated polymer by pyrogenic lipopolysaccharides present in the cell wall of gram-negative microorganisms is eliminated. The bacterial strain Tepidimonas taiwanensis LMG 22826T was able to produce high yields of biomass and PHA on a mixture of glucose and fructose substrate. Using grape pomace extract, almost identical values to those obtained on pure substrates were achieved. The thermophile Caldimonas thermodepolymerans DSM 15344, originally named Schlegelella thermodepolymerans, is a very promising PHA producer on xylose-based substrates. These substrates can be, for example, hydrolysates of lignocellulosic materials, which represent a sustainable source of carbon. Their suitability for the cultivation of C. thermodepolymerans was tested on model hydrolysates composed of pure carbohydrates. To approximate real samples, the effect of potential microbial inhibitors present in lignocellulosic hydrolysates was also investigated. An innovative isolation protocol for the recovery of PHAs based on osmotic stressing of thermophilic and halophilic microorganisms under elevated temperature with the addition of a low concentration surfactant solution was also developed as part of the thesis.
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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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