National Repository of Grey Literature 97 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Evolutionary engineering of PHA producing bacterium Halomonas halophila
Ikrényiová, Terézia ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with evolutionary engineering of PHA producing bacteria, the principle of PHA production and solution of this production’s disadvantages, but also applications of these biopolymers in the theoretical part. The production of polyhydroxyalkanoates by bacteria Halomonas halophila, which is focused on gaining the maximus amount of 3-hydroxyvalerate in formed copolymer, is described in the experimental part. The precursor valeric acid was added to bacteria due to gain the amount of 3HV. It was found that the concentrations over 3 g/l aren´t usable for production sufficient concentration of PHA. The very low concentrations of valeric acid led to low amount of 3HV in PHA. The available concentration of this precursor for production sufficient concentration of PHA by bacterial cell is 3 g/l. Moreover, it was found that the valeric acid should be added after 24 hours of cultivation in mineral production medium. The thesis is also concentred on comparison the original bacterial strains of Halomonas halophila to strains, which were adapted on valeric acid as stress factor for bacteria. The assumption, that the adapted strains can better utilize valeric acid and the incorporation to copolymer of it is higher like the original strains, was affirmed.
Isolation of PHA producing bacteria from mixed microbial consortia
Plachý, Petr ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is detection of polyhydroxyalcanoates (PHA) producing bacteria from activated sludge and effort for isolation of these bacteria. The theoretical part deals with general issues of PHA and of bacterial production of PHA. Also there is attention paid to characterization of activated sludge and to selected methods used in this thesis for detection of PHA producing bacteria. In the experimental part, mixed microbial culture of the activated sludge was cultivated on different carbon sources. Potential PHA producers was isolated from these cultures with the use of lipophilic staining with Nile red and phaC gene (essential for PHA synthesis) was detected with the use of polymerase chain reaction (PCR) in 11 of the isolated cultures. By DNA sequencing 8 bacterial cultures were identified. It was Klebsiella pneumoniae (1 x), Paenirhodobacter enshiensis (1 x) and Pseudomonas putida (6 x). Presence of PHA in biomass was detected in 2 of the 11 isolated cultures by Fourier transformation infrared spectroscopy (FTIR) analysis. The content of polyhydroxybutyrate (PHB) was determined with the use of gas chromatography to 9,33 % of dry biomass (Paenirhodobacter enshiensis) and to 1,18 % (unidentified culture).
Evaluation of grape pomace as carbon source for bacterial cellulose production
Stříž, Radim ; Obruča, Stanislav (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis is focused on the production of bacterial cellulose by Gluconacetobacter xylinus. The theoretical part consists of a brief description of G. xylinus, properties and uses of bacterial cellulose. Firstly, the experimental part focuses on optimization of production of bacterial cellulose by G. xylinus CCM 4611. This optimization was focused on pH value, a cultivation type, and used carbon source for production. The optimal pH was 6.5. The carbon source showing the highest production of bacterial cellulose was mannose for the dynamic production and saccharose for the static production of bacterial cellulose. The second part of the experimental part focuses on a comparison of two methods for assaying reducing sugars – Somogyi-Nelson (SN) assay and dinitrosalicylic acid (DNS) assay. The comparison of two above mentioned methods showed that the results obtained by the SN method were very close to High performance liquid chromatography (HPLC) data. It can be concluded that HPLC method is the best method if we need to know the type of sugars but if we need only the quick determination of the sum of the reduction sugars, SN method is very suitable. 4 It can be concluded that the Moravian region produces a large amount of winery biomass. Therefore, the use of the pomace extract as a carbon source for the production of bacterial cellulose seems to be interesting mainly from the point of the recirculation economy. The obtained results showed that the grape sugar extract could be used, and from the production values, it is even more suitable for the production of bacterial cellulose than industrial sugars.
Use of Molecular Biology Techniques for Identification and Analysis of Probiotic Bacteria
Konečná, Jana ; Doškař, Jiří (referee) ; Kráčmar, Stanislav (referee) ; Obruča, Stanislav (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
The effects of chemicals on cell lines viability
Zemanová, Anita ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
The subject of this diploma thesis is the influence of selected chemicals on cell lines viability. The theoretical part contains options of cancer treatment by using chemotherapeutics including their mechanism of action and side effects. Additionally, there are described alternative DNA structures with focus on G-quadruplexes and ligands that interact with G-quadruplexes. These compounds are promising drugs in cancer treatment due to their high specificity to G-quadruplexes, which are found in telomeres of chromosomes. G-quadruplex interacting ligands by stabilization of G-quadruplexes can inhibit the enzyme telomerase, which is necessary for telomere lengthening of rapidly dividing cancer cells. Additionally, the possibilities of viability assays are summarized in the theoretical part. The aim of the experimental part was comparing cytotoxic activity between commercially available chemotherapeutics and selected G-quadruplex interacting ligands. Another task was the study of apoptosis and necrosis after the treatment of selected chemicals on cell lines and after the localization of ligands interacting with G-quadruplexes in the cells of the breast cancer cell line. In the experimental part, G-quadruplex interacting ligands have been shown to exhibit similar cytotoxic activity to commercially available chemotherapeutic agents.
Biotechnological production of sophorolipids
Šimšová, Veronika ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the microbial production of sophorolipids by the Starmerella genus yeasts. The theoretical part of the thesis includes general characteristics of biosurfactants with the focus on sophorolipids. There are described the options of biotechnological production of sophorolipids and fields of their possible applications. Furthermore, the theoretical part deals with the describtion of Starmerella bombicola yeast, which is often used for biotechnological production of sophorolipids. Six strains of the Starmerella genus were cultured in the basic medium suitable for sophorolipid produsction. The amount of produced sophorolipids was tested by the emulsification capacity assay, solubilization of crystalline anthracene assay, measuring the surface tension by the Du-Noüy-Ring method and determination of the sophorolipid concentration by extraction with ethyl acetate. The purity of the extracted sophorolipids was inspected by the Fourier Transform infrared spectrosopy (FT-IR) Based on the results, the Starmerella bombicola CBS 6009 and the Starmerella anomalae CBS 14178 strains were studied in greater detail. They were cultured in several production media of different composition and the effect of the individual components on the ability of the sophorolipid production was monitored. Based on the results, it was evaluated that the composition of the medium has a great influence on the production ability of the strains. The highest production rate of sophorolipids was achieved in the basic production medium and so was in the medium containing molasses and Indian waste oil. The cultivation mode has great effect on the sophotolipid production rate. It has been found that when cultured in a bioreactor, the strains produced sophorolipids in a larger amount and of a different quality than in the shaker flasks.
Study on potential applications of glutamic acid polymer
Čangelová, Katarína ; Matoušková, Petra (referee) ; Obruča, Stanislav (advisor)
The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.
The effect of magnetic field and other selected stressors on physiology of bacterial cells
Mrázová, Kateřina ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the effect of magnetic field and organic substances, namely benzene and p-nitrophenol, on cell of PHA producing bacteria Cupriavidus necator H16 and mutant strain Cupriavidus necator PHB4, which does not produce polyhydroxyalkanoates. Static magnetic field was generated by both permanent magnet and electromagnet. The effect of magnetic field on the growth of bacterial cells was studied using growth curves. It was found that cultivation in magnetic field and mineral medium mostly inhibits bacterial growth. Also the amount of polyhydroxyalkanoates was observed using FT-IR, flow cytometry and microscopy with fluorescent dye. Growth curves and flow cytometry were also used to study the influence of organic substances on bacterial cells. It was found that while benzene does not affect either C. necator H16 or C. necator PHB4, p-nitrophenol acts as the inhibitor of bacterial growth for both cultures. Finally the impact of p-nitrophenol on the accumulation of PHA was studied using gas chromatography.
Screening of biotechnological potential of selected members of the genus Geobacillus and other related genuses
Kouřilová, Xenie ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.
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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