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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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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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Regulation of polyhydroxybutyrate production in Wautersia eutropha
Grossová, Marie ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
The aim of this work is study of microbial production of biodegradable polymers (polyhydroxyalkanoates; polyhydroxybutyrate PHB). Bacterium Wautersia eutropha was used for laboratory production of PHB. In this bacterium application of exogenous stress factors (osmotic shock, hydrogen peroxide and ethanol) was tested to enhance PHB production. Influence of exogenous stress on bacterial culture depended on stress factor concentration and time of stress application. Substantial influence on PHB yield exhibited changes of biomass production during stress experiments. The main part of this work is focused on study of PHB production in microbial cells exposed to stress in different growth phases. Application of strong stress in early phases of growth led to PHB and biomass decrease. The most suitable seems to be application of stress at the beginning of stationary phase accompanied by increase of PHB formation. Concentration of stress factor could be optimized too. The highest yields were obtained after addition of 0.5 % ethanol into production medium at 60th hour (PHB increase 21 %). These results can form a basis for new fermentation strategy which leads to enhanced effectiveness of PHB production.
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Behaviour of microorganisms using microcalorimetry
Piletskaya, Maryia ; Sedláček, Petr (referee) ; Krouská, Jitka (advisor)
The diploma thesis deals with the study of the behavior of a microorganism with the help of microcalorimetry. The theoretical part of the thesis deals mainly with the characterization of the microorganism Rhodospirillum rubrum selected for experimental work and the application of microcalorimetry in microbiology. The second goal was to screen the growth of the bacterial strain R. rubrum using different substrates, and subsequently, based on experimental results, to select the most suitable substrate, and optimize its concentration and aeration of the medium for the microorganism using microcalorimetry. Hexanoate, malate, acetate and fructose were used to examine the most suitable substrate by microcalorimetry. Another goal was data processing and discussion of the advantages and disadvantages of substrate selection and media aeration with respect to bacterial strain growth. Hexanoate was found to be the best substrate for growth, followed by optimization of the concentration and aeration of the medium. The results of the microcalorimetric measurement were confirmed by spectrophotometric study of the growth curve of the investigated bacteria. The effect of osmotic pressure on bacteria and the dependence of their survival on the amount of polyhydroxyalkanoates (PHA) in the cell were also studied. The amount of PHA in the biomass was determined chromatographically.
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Utilization of selected extremophilic bacteria for bioplastic production
Vidláková, Michaela ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on the study of the biotechnological potential of thermophilic bacteria Tepidiphilus thermophilus, Tepidimonas taiwanensis and Tepidimonas fonticaldi. The aim of this work was to investigate the ability of bacteria to produce polyhydroxyalkanoates (PHA). The theoretical part includes the characteristics of PHA and extremophilic bacteria. In the experimental part, the investigated bacteria were tested by molecular technique PCR for the presence of the phaC gene encoding for the first class PHA synthase. The presence of the phaC gene was confirmed in bacterial strains Tepidiphilus thermophilus and Tepidimonas taiwanensis. The ability of PHA production on various carbon sources was examined for all the selected microorganisms, optimal temperatures for cultivation were determined, and the ability to synthesize copolymers was investigated by adding suitable precursors to the production medium. The most intensive PHA production was recorded in the bacterial strain Tepidimonas taiwanensis, but other strains were also able to produce PHA. The ability to produce P(3HB-co-3HV) or P(3HB-co-4HB) copolymers has also been demonstrated in some strains. A significant amount of P(3HB-co-3HV) was obtained using the Tepidimonas taiwanensis strain. Morever, Tepidimonas taiwanensis strain was also tested for its ability to use various model lignocellulose hydrolysates to form PHA. The highest PHA gain was obtained using model hydrolysate of softwood. Furthermore, the ability of proteases production by selected bacterial strains was tested. The presence of these enzymes was detected in the Tepidimonas taiwanensis strain, which was followed by determination of the temperature optima of the proteases, the pH optima of the proteases and also of their molecular weights.
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Study on metabolism of polyhydroxyalkanoates in Rhodospirillum rubrum
Rubanová, Blanka ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoate (PHA) metabolism in Rhodospirillum rubrum under different cultivation conditions. The conditions differed in the aeration rate (aerobic, anaerobic, microaerobic) and in the presence of a light source (light cultivation, dark cultivation). First, different volumes of medium were suggested and tested for the fastest and the most suitable inoculum cultivation. Subsequently, cultures of Rhodospirillum rubrum under different conditions in terms of PHA content were compared by gas chromatography. In addition to gas chromatography, other complementary methods were used for better characterization of bacterial growth and metabolism, such as flow cytometry, UV-VIS spectroscopy (transmission and diffusion transmission modes). Fluorescence microscopy and cryo-scanning electron microscopy were used in terms of cell morphology. Furthermore, the ability of bacteria to synthesize color pigments (carotenoids and bacteriochlorophyll a) was investigated. The bacteria were cultivated on different types of media (liquid, solid), the color pigments were subsequently extracted, and their concentrations were calculated. UV-VIS spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) were also used to measure color pigments.
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Biotechnological production of PHA copolymers containing 4-hydroxybutyrate
Kovářová, Radka ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.
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Isolation, identification and characterization of extremophiles capable of PHA production
Vlasáková, Terézia ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.
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Study on metabolic properties of the bacterium Schlegelella thermodepolymerans with respect to biosynthesis of polyhydroxyalkanoates
Chvátalová, Viktorie ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This bachelor's thesis deals with the metabolic properties of the thermophilic bacterium Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates (PHA). The aim of the study was to understand the metabolism and utilization of different carbohydrates (xylose, cellobiose, glucose and their combinations) depending on the cultivation time. In the experimental part, two bacterial strains (M15344 and DSM15344) were used. The increase in optical density was monitored during the measurements for the bacteria. Furthermore, the content of P(3HB) from the biomass was determined using GC-FID and the molecular weight of the polymer was determined using SEC- MALS analysis. By measuring residual sugar using HPLC-RI, the course of carbohydrate utilization was determined as a function of time. The results of both bacteria were compared and evaluated. DSM15344 showed better results in P(3HB) production than M15344, and cellobiose and xylose appeared to be the most suitable substrates for P(3HB) production.
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Production of polyhydroxyalkanoates by bacterium Schlegelella thermodepolymerans
Černý, Vojtěch ; Černayová, Diana (referee) ; Pernicová, Iva (advisor)
The aim of this work was to study capability of various strains of thermophilic bacteria Schlegelella thermodepolymerans incorporate diverse monomer units to structure of polyhydroxyalkanoates (PHA). Precursors for production mcl-PHA, 4-hydroxybutyrate and 3-hydroxyvalerate were tested. The theoretical part deals with characteristic of studied polymers, properties and applications and usage of thermophilic bacteria in industry. In experimental part there were undergone cultivations with different precursors, which were added in different times and concentrations. Four bacterial strains S. thermodepolymerans from various public collections of microorganisms were used during these cultivations. Levulinic acid, added at start of cultivation, was chosen as the best precursor. Afterwards there was cultivation with concentration line to find out, whether concentration influence composition of copolymer. Increasing the amount of precursor led to decreasing quantity of biomass and PHA, but there were very interesting materials with high amount of 3HV. Addition 8 g/l of precursor led up to 52 %. Subsequently there was found out molecular height of polymers by size-excluion chromatography (SEC) combined with multi-angle light scattering (MALS).
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