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Isolation of bacterial flagellin as an immunogen for the preparation of antibodies for cystic fibrosis patients
Antipova, Valeriia ; Hodek, Petr (advisor) ; Vyhnalová, Kateřina (referee)
Cystic fibrosis (CF) is an autosomal recessive disease affecting mainly the lungs, but also other organs including the liver, pancreas and reproductive organs. In the affected organs, there is a build-up of thick sputum called mucus. CF is caused by mutations in the CFTR gene, which is located on the long arm of the 7. chromosome. These mutations then lead to mutations in the encoded CFTR protein. Mutations in the CFTR protein cause defects in chloride ion transport. One of the most common symptoms of CF is frequent lung infections caused by bacteria that multiply rapidly in the lung mucus. One of those bacteria is Burkholderia cepacia (BC). BC is an aerobic, gram-negative, conditionally pathogenic microorganism that poses a danger to immunocompromised people. BC and some other microorganisms have been called "hospital- acquired bacteria", It is a problem for CF patients who are often hospitalized. BC most often contain a single polar flagellum that serves to move and adhere to the surface of host cells. The flagellum, which is primarily composed of the flagellin protein, represents one of the main virulence factors of BC. The purpose of this research is to find an optimal method for isolating bacterial flagellin from Burkholderia cepacia. Flagellin can then serve as an immunogen for the...
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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 Raman spectroscopy and Raman tweezers for analysis and isolation of PHA producing bacteria
Beránková, Barbora ; Enev, Vojtěch (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the study of the utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of polyhydroxyalkanoates (PHA) producing bacteria. Using gas chromatography with FID detection, we determined the polyhydroxybutyrate (P(3HB)) content of the PHA biomass of bacterial strains Burkholderia cepacia, Halomonas halophila, Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 and Lactobacillus delbrueckii, which is not a producer of polyhydroxyalkanoates but this bactrea was selected as representative of Gram-positive bacteria. Subsequently, thanks to Raman microspectroscopy, Raman tweezers and FT-IR spectrometer in combination with Raman FT-module, we were able to confirm or disprove the presence of P(3HB) in bacteria. Furthermore, the thesis describes Cupriavidus necator H16, which is a model organism for the production of P(3HB), and his mutant strain Cupriavidus necator H16/PHB-4. The bacterial strain Cupriavidus necator H16 was cultivated in a production mineral medium of various nitrogen contents, while cultivation was also carried out in liquid Nutrient Broth. By this cultivation we were able to reach various P(3HB) content in bacterial biomass, the spectra were subsequently compared with the spectrum of the bacterial strain Cupriavidus necator H16/PHB-4. Raman spectroscopy is well used to characterize the composition of individual bacterial cells, is a fast, versatile, and virtually non-invasive tool for studying cells.
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Study on pretreatment and hydrolysis of selected lignocellulose materials
Kovářová, Markéta ; Dvořák, Miloš (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on study of chemical and enzymatical hydrolysis of raw wood material. The aim of this work was to find the suitable method for pretreatment of selected lignocellulose materials. The theoretical part deals with characterization of lignocellulosic material and its components. There are also subscribed various pretreatment methods and their effect on hydrolysis of sawdust. In experimental part of the work the most appropriate approach of pretreatment and hydrolysis of sawdust was studied. Criteria for the selection of suitable method was concentration of saccharides as desired product of hydrolysis and also concentration of the most important microbial inhibitors - polyphenols. Application of 96% ethanol or 5% H2O2 were identified as the most promising pretreatment methods which enhanced yields of fermentable sugars about 30 %. Further, we also performed cultivation of bacteria Burkholderia cepacia and bacteria Burkholderia sacchari using solution obtained by hydrolysis of lignocellulose material.
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Enzymatic hydrolysis of wastes associated with coffee production
Kovářová, Markéta ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of potential production of extracellular hydrolytic enzymes by microorganisms – bacterium and moulds, which have been cultivated on spent coffee grounds. The theoretical part deals with characterization of coffee and utilization of coffee by-products. There are also subscribed microorganisms and enzymes which have been noticed. In experimental part coffee ground was used as the sole substrate for production of extracellular hydrolytic enzymes. Productions of protease, cellulase, mannase and lipase enzymes were observed. None-identified isolate of mould spontaneously contaminating spent coffee grounds was identified as the best producer of these enzymes. Subsequently the conditions of cultivation such as water content and shaking vs. static cultivation of this moulds were optimized. Further, we performed partial purification and pre-concentration of the enzyme cocktail by ultrafiltration, ultradialysis and PAGE-SDS characterization of extracellular enzymes was performed as well.
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Detoxification of lignocellulose hydrolyzates
Vašíčková, Monika ; Pořízka, Jaromír (referee) ; Obruča, Stanislav (advisor)
The aim of this work was study of the detoxification of lignocellulose material hydrolysates and to investigate sawdust suitability as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most suitable way of detoxification of model and real hydrolysate was studied. After that, detoxification methods used were evaluated. Criteria for evaluation were concentration of polyphenols as the most important microbial inhibitors and reduction saccharides as the main carbon substrate. Furthermore, fermentability of the hydrolysates was also tested by cultivation of two bacteria capable of PHA accumulation. Burkholderia sacchari demonstrated higher ability to accumulate PHA then Burkholderia cepacia. Then in the summary – most effective way for detoxification was ‚overliming‘. Major increase of PHB in biomass was obtained when Burkholderia sacchari was cultivated on media gained by application of overliming of real lignocellulose hydrolysate. However, total gains of PHB were more likely low and then sawdust can not be considered as a substrate for PHB production at industrial scale.
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Polyhydroxyalkanoates and their role in bacterial biofilms
Rucká, Markéta ; Samek,, Ota (referee) ; Obruča, Stanislav (advisor)
This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.
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Experimental study on the adsorption of bacterial cells on solid surfaces
Kahanovská, Kristína ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This diploma thesis focuses on an optimalization of simple laboratory model systems which serve as an innovative tool for an experimental study on the adsorption of bacterial cells on solid surfaces. In the description of living biological systems, an adsorption is labelled as an adhesion. Designed model systems were validated with a physical-chemical analysis. Various techniques were used to determine bacteria properties, more specifically Burkholderia cepacia and Bacillus megaterium. The solid surfaces after sorption of bacterial cells of Bacillus megaterium were subjected to a structural and visual analysis. Applying the theoretical approach (e.g. using different physical-chemical models) to study the adhesion of microorganisms to a particular surface allows a prediction of the conditions for a successful adhesion. The results will give us a better understanding of a formation and development of a biofilm.
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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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Utilization of spent wood chips for biotechnological production of PHA
Ladický, Peter ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the suitability of wood shavings and sawdust as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most appropriate approach of hydrolysis of wood shaving and sawdust and the effect of polyphenol and furfural concentration on ability to accumulate PHA was studied. Burkholderia sacchari had greater ability to accumulate PHA compared to Burkholderia cepacia. PHB values 87–89 % were achieved when Bulkholderia sacchari was cultivated on medium that contained detoxified hydrolysate of sawdust. Sawdust is therefore a promising substrate for microbial production of PHA in terms of reducing production costs and high content of PHB in biomass.
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