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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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Analysis of bacrerial cells employing flow cytometry and flurescence microscopy
Müllerová, Lucie ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.
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Study on PHA production by selected thermophillic bacteria
Brondová, Zuzana ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of thermophilic bacterial strains Tepidiphilus thermophilus and Chelatococcus daeguensis aimed at the production of polyhydroxyalkanoates (PHA). Experiments were developed to determine properties of use of these microorganisms in the biotechnological industry. In the experimental parts, the presence of the phaC gene indicating PHA production was confirmed by both bacteriological strains by polymerase chain reaction (PCR). Subsequently, the conditions for PHA production were optimized. Condition profiling aimed at determining the optimal temperature, optimal carbon source, and the effect of the precursor on cell growth, composition and amount of PHA through GC-FID. The last experiment was to determine the utilization of selected substrates. The optimal conditions for the Tepidiphilus thermophilus strain were temperature above 55 ° C and glycerol as the best carbon source. Precursors inhibited cell growth, but the highest amount of 3HV was determined when valeric acid was added to medium. The Chelatococcus daeguensis strain had an optimal temperature of 45 ° C and glucose was optimal carbon source. The best cell metabolizing precursor was 1,4-butanediol. Both bacterial strains were capable of utilizing many substrates where glucose and valeric acid were best.
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Microbial production of extracellular polymers and their involvement in stress response
Müllerová, Lucie ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
Bachelor’s thesis is focused on the production of extracellular polysaccharide pullulan by microorganism Aureobasidium pullulans, purification of pullulan and its possible use as a cryoprotectant. As a part of this work a description of A. pullulans, pullulan and an overview of involvement in its stress response were provided. In the experimental part growth characteristics of A. pullulans (the strain CCM 8182 was used) and pullulan production during growth in optimal conditions was analyzed. Biomass production was the highest with fructose as a carbon source (3,580 g/l CCM 8182), the highest pullulan production occurred when using sucrose as a carbon source (10,300 g/l F 148). Among three organic solvents used for pullulan precipitation – ethanol, acetone, isopropylalcohol, ethanol was selected as the best for further experiments in ratio 1:2 (fermentation broth:ethanol). Pullulan purity was characterized by HPLC. As a further part of this work, cryoprotectant properties of pullulan at temperatures – 72° C, -18° C, 4° C and 60°C were studied. The presence of pullulan at temperatures – 72° C and 60° C was found to be detrimental to cell viability. At temperatures – 18° C and 4° C the cryoptotectant activity of the polysaccharide was confirmed.
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Biotechnological production of PHA employing selected extremophiles
Kurzová, Pavlína ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of production of polyhydroxyalkanoates (PHA) by bacteria Halomonas salina (CCM 4361T). Theoretical part deals with the properties and biosynthesis of PHA and chracterization of extremophiles bacteria. The experimental part is focused on cultivation of bacteria on different substrates and concentration of sodium chloride in medium for optimal production of PHA. Content of biomass and PHA were analyzed by spektrofotometry and gass chromatography with FID. The fructose proved to be the most suitable substrate for a production of PHA, with a content of 12,55 % of PHA in biomass. Optimal concetration of NaCl was found at 80 g/l. Bacteria were investigated by molecular technique polymerase chain reaction (PCR) and detected by gel elctrophoresis. Test confrimed opresence of phaC gene encoding for PHA synthase, enzyme which is necessary for PHA biosynthesis.
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Thermophillic producers of PHA
Hollá, Tereza ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor’s thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Chelatococcus thermostellatus. Polyhydroxyalkanoates are natural biopolymers produced by microorganisms. Microorganisms use them to store energy and carbon under stress conditions. In experimental part of this work the most appropriate conditions for growth of biomass and polyhydroxyalkanoates accumulation were detected. The capability of copolymer P(3HB-co-3HV) production by bacterial strain was also studied. Glucose was determined as the most appropriate carbon source for biomass growth and P(3HB) production. Content of P(3HB) in biomass was 59,42 % per cell weight. The accumulation of the copolymer by bacterial strain was detected, but at very low concentrations.
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Utilisation of fluorescence techniques for analysis of industrially relevant microorganisms
Müllerová, Lucie ; Kráčmar, Stanislav (referee) ; Krčma, František (referee) ; Obruča, Stanislav (advisor)
Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodidem. Tento protokol byl také využit pro studium morfologie PHA-syntetizující halofilní bakterie Halomonas halophila. Pro stanovení UV-protektivních vlastností PHA obsaženého v bakterii Cupriavidus necator, byl optimalizován další protokol, tentokrát pro ROS citlivou sondu CM-H2DCFDA, která byla stanovená jako citlivější v porovnání s její nemethylovanou formou. V této studii byly navíc potvrzeny UV-protektivní vlastnosti PHA na bakterii Cupriavidus necator H16 v porovnání s PHA nesyntetizujícím kmenem Cupriavidus necator PHB-4.
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The forms of parents appraisal and their influence on self-efficacy of preschool child
Müllerová, Lucie ; Horáková Hoskovcová, Simona (advisor) ; Mertin, Václav (referee)
The theoretical part of the thesis deals at first with the theoretical principles of self-efficacy. The subsequent part treats of the developmental characteristics of preschool stage with a view to connection with the parents appraisal. The final part aims at appraisal, in term of general approach to the child, in term of rewards and punishments and in term of the appraisal as a formalizing agent in the formation of the basics of self-efficacy. The empirical part presents a research on forms of mothers appraisal on children accomplishments and failures while performing a specific task. The quantitative analysis traces appearance of forms of appraisal and their influence on child selfefficacy. The qualitative part is focusing especially on mothers non-appraising reactions on children while performing a specific task.
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Term of evolution and its understanding by students of secondary schools and high schools.
Müllerová, Lucie ; Teodoridis, Vasilis (advisor) ; Pavlasová, Lenka (referee)
The term of evolution and its understanding by students of secondary schools and high schools This Master's thesis deals with the conception of evolutionary topic in schools and textbooks. It introduces education systems and curricular documents of England, Scotland and the Czech Republic. Based on this information an analysis of biology textbooks was made, which was focused on the theme of evolution. The research included 62 textbooks distributed by 27 different publishers. Comparative SWOT analysis of these textbooks has shown that the concept of biology in general is presented differently, what is also reflected in the interpretation of the studies of evolution. The questionnaire research based on those findings was done in the Czech Republic. It is focused on secondary school and high school pupil's knowledge and their personal opinions about this topic. 586 pupils took part in this research. It was found that in the context of evolutionary processes the pupils have problems to use commonly known biological, or evolutionary terms (e.g. adaptation, evolution of the organisms). On the other hand some pupils can easily define some more complicated evolutionary topics such as Neo-Darwinism. Pupils have quite various personal opinions on the issue of the evolution. Some of them are not interested in...
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