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Biopolymers produced by halophillic bacteria and their role in stress response
Drábková, Kateřina ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
The subject of this thesis is the study of halophilic microorganisms. The main part deals with isolation and characterizatin of the extracellular polysaccharide (EPS) produced by the archeal strain Haloferax mediteranei. Chemical structure was determined by Raman spectroscopy, FTIR and elementar analysis. Very low amount of sulphate groups indicate, that generally accepted structure of the EPS is not universal and can be dependent upon cultivation condition. Further, EPS revealed substantial protective effect for both model prokaryotic organisms – Cupriavidus necator and model eukarytoic microbe – Saccharomyces cerevisiae when the cells were exposed to various stress factors such as freezing, high temperature, osmotic pressure or ethanol.
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Hydrogen production by bacteria of genus Clostridium
Sedláček, Zbyněk ; Turková, Kristýna (referee) ; Rittich, Bohuslav (advisor)
The bachelor thesis deals hydrogen production by bacteria of the genus Clostridium. Thesis gives an overview about hydrogen production mentioned microoganism, characterized further biotechnological methods and show examples of industrial production of hydrogen. Also there are describes the quantitative characteristic and metabolism of bacteria genus Clostridium and examples of usable substrates. Polymerase chain reaction (PCR) is used to the detection and identification by species and specific primers. DNA isolated by fenol extraction from bacterial culture Clostridium tyrobutyricum DSM 2637 was amplified using genus-specific PCR to form PCR products size 619 bp.
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Study of Probiotic Lactic Acid Bacteria Producing Antimicrobial Compounds
Turková, Kristýna ; Doležal, Petr (referee) ; Rada,, Vojtěch (referee) ; Španová, Alena (advisor)
The sixty-eight strains isolated from breastfed full-term infant feces and from another sources were identified using genus-specific polymerase chain reaction (PCR) for Lactobacillus, species-specific PCRs, multiplex PCR, pheS PCR, rep-PCR, RAPD-PCR and 16S rDNA sequencing into Lactobacillus species or group of species. Seven strains produced antimicrobial proteinaceous substances in the supernatants. Antimicrobial proteinaceous substances of three strains were tested on temperature, pH a detergent stability. All tested strains produced temperature-stable antimicrobial proteinaceous substances. Antimicrobial activity was not influenced by detergents with exception of SDS. Presence of genes for production of bacteriocins (acidocin B, gassericin A, gassericin T, gassericin K7A and gassericin K7B) were detected in DNA of fourteen strains using PCR and DNA/DNA hybridization. Selected PCR products were sequenced and analyzed using BLAST algorithm and CLUSTAL W2 programme. The sequences of specific PCR products in DNA of two strains had 100% similarity with the sequences from the database GeneBank. Selected strains of Lactobacillus acidophilus group were tested for the surveillance in gastrointestinal tract, for the production of antimicrobial substances, for the adhesion on Caco-2 cells and for the presence of genes of antibiotic resistance. DNA of strains was tested using specific primers on the presence of genes for histidine-decarboxylase, tyrosine-decyrboxylase and linoleate isomerase. The gene for histidine-decarboxylase production was detected in DNA of seven strains, for tyrosine-decarboxylase production in DNA of one strain and for linoleate isomerase in DNA of four strains. Imunomagnetic separation of the cells was optimized. Magnetic particles functionalized with streptavidin and the anti-Lactobacillus antidote was used for the separation of the cells of Lactobacillus rhamnosus LOCK 0900 from MRS medium, UHT milk and from the yogurt. The IMS-PCR was used for detection of imunomagnetic separated bacterial cells.
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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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Methods for identification of PHA producing bacteria
Skřivanová, Veronika ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.
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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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Biopolymers produced by halophillic bacteria and their role in stress response
Drábková, Kateřina ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
The subject of this thesis is the study of halophilic microorganisms. The main part deals with isolation and characterizatin of the extracellular polysaccharide (EPS) produced by the archeal strain Haloferax mediteranei. Chemical structure was determined by Raman spectroscopy, FTIR and elementar analysis. Very low amount of sulphate groups indicate, that generally accepted structure of the EPS is not universal and can be dependent upon cultivation condition. Further, EPS revealed substantial protective effect for both model prokaryotic organisms – Cupriavidus necator and model eukarytoic microbe – Saccharomyces cerevisiae when the cells were exposed to various stress factors such as freezing, high temperature, osmotic pressure or ethanol.
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Publishing house Fr. A. Urbánek archives
Turková, Kristýna ; Gabrielová, Jarmila (referee) ; Ottlová, Marta (advisor)
The main part of the diploma thesis is comprised of a catalogue of archived fragments from the Fr. A. Urbánek Publishing House situated within the library of the Institute of Musicology, Faculty of Arts, Charles University in Prague. The textual part contains two general chapters, approaching both contemporary music publishing and the Fr. A. Urbánek Publishing House. The third chapter goes on to evaluate and compare the fragment from the archives of the Fr. A. Urbánek Publishing House with archived resources housed in other Prague-based institutions. The aim is to define the range of sources that could have been part of the aforementioned original archives of the publisher.
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Methods for identification of PHA producing bacteria
Skřivanová, Veronika ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.
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Study of Probiotic Lactic Acid Bacteria Producing Antimicrobial Compounds
Turková, Kristýna ; Doležal, Petr (referee) ; Rada,, Vojtěch (referee) ; Španová, Alena (advisor)
The sixty-eight strains isolated from breastfed full-term infant feces and from another sources were identified using genus-specific polymerase chain reaction (PCR) for Lactobacillus, species-specific PCRs, multiplex PCR, pheS PCR, rep-PCR, RAPD-PCR and 16S rDNA sequencing into Lactobacillus species or group of species. Seven strains produced antimicrobial proteinaceous substances in the supernatants. Antimicrobial proteinaceous substances of three strains were tested on temperature, pH a detergent stability. All tested strains produced temperature-stable antimicrobial proteinaceous substances. Antimicrobial activity was not influenced by detergents with exception of SDS. Presence of genes for production of bacteriocins (acidocin B, gassericin A, gassericin T, gassericin K7A and gassericin K7B) were detected in DNA of fourteen strains using PCR and DNA/DNA hybridization. Selected PCR products were sequenced and analyzed using BLAST algorithm and CLUSTAL W2 programme. The sequences of specific PCR products in DNA of two strains had 100% similarity with the sequences from the database GeneBank. Selected strains of Lactobacillus acidophilus group were tested for the surveillance in gastrointestinal tract, for the production of antimicrobial substances, for the adhesion on Caco-2 cells and for the presence of genes of antibiotic resistance. DNA of strains was tested using specific primers on the presence of genes for histidine-decarboxylase, tyrosine-decyrboxylase and linoleate isomerase. The gene for histidine-decarboxylase production was detected in DNA of seven strains, for tyrosine-decarboxylase production in DNA of one strain and for linoleate isomerase in DNA of four strains. Imunomagnetic separation of the cells was optimized. Magnetic particles functionalized with streptavidin and the anti-Lactobacillus antidote was used for the separation of the cells of Lactobacillus rhamnosus LOCK 0900 from MRS medium, UHT milk and from the yogurt. The IMS-PCR was used for detection of imunomagnetic separated bacterial cells.
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