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Raman spectroscopy as a tool for analysis of microbial cells
Pokorný, Petr ; Enev, Vojtěch (referee) ; Samek, Ota (advisor)
This bachelor thesis is focused on the use of Raman spectroscopy for analysis of ectoine and hydroxyectoine presence in bacterial strain Halomonas elongata. Theoretical part compile characteristics of extremophiles with closer look on halophilic organisms and one of their main osmolytes, ectoines. Following by description of Raman spectroscopy method and its uses. Experimental part deals with cultivation of bacteria on different combinations of substrate and salt concentration in order to reach optimal production of PHA and for achieving the highest possible yield of biomass to be analysed by Raman spektroskopy. As the best substrate for PHA production turned out to be glukose along with salt concentration 30 g/l NaCl, where percentage representation of PHB makes 30,5229 %. As an ideal substrate for the highest yield of biomass proved to be sacharose. Bacteria that grew on sacharose with three different salt concentrations, 40, 70 and 100 g/l NaCl was measured along with Halomonas salina and Halomonas organivorans for comparison. Raman spectroscopy unfortunately turned out to be inadequate for measurement of this type of sample, thus we were not able to measure ectoine of hydroxyectoine presence in cells.
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Experimental study on the release of active ingredients from gel matrices
Volhinava, Anastasiya ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
This thesis deals with the characterization of hydrogels containing humic substances and salicylic acid. Experimental study of the transport of active substances was performed from the gel matrix over the skin and synthetic membranes. During this work was writing literature review focused on the use of gels with humic substances in cosmetic, medical and pharmaceutical industries. The model humic hydrogels was prepared with salicylic acid and their characterization by basic methods of material analysis. These basic methods were and visual assessment of consistency during the preparation and rheology. The study of the release of active and humic substances was performed due to the vertical diffusion cells. The amount of released humic substance was characterized by UV-VIS method and the amount of released salicylic acid from gel matrix was determined by HPLC.
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Utilization of vibrational spectroscopy for in-situ qualitative and quantitative analysis of polyhydroxyalkanoates in microbial biomass
Kevélyová, Barbora ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis was to evaluate the applicability of selected methods of vibrational spectroscopy for qualitative and quantitative analysis of biomass, which contains polyhydroxyalkanoates. Fourier-transform infrared spectroscopy, that uses attenuated total reflection technique (ATR-FTIR), was chosen for this experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) has been studied in Cupriavidus malaysiensis, Thermomonas hydrothermalis and Aneurinibacillus thermoaerophilus sp. H1. Data were obtained by evaluating the infrared spectra, which were then compared with the results of performed gas chromatography. Visual changes in the spectra were also observed along with crystallinity. No correlation was detected between the data obtained by gas chromatography and by the method ATR-FTIR in quantitative analysis. Thus, this method is not suitable for determining the content of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in biomass in the set experiment and the bacteria used. On the other hand, the method was successfully used for in-situ qualitative analysis of copolymer in the samples of Aneurinibacillus thermoaerophilus sp. H1. The degree of data correlation was relatively high in the given experiment. With these findings, ATR-FTIR is a suitable analytical method for determining the monomer composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in this strain. Comparison with the PHA producer Cupriavidus malaysiensis showed that this method is not universal for all types of microorganisms observed in this thesis.
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Study of humic and fullvic acids by excitation emission matrix method
Enev, Vojtěch ; Vala, Martin (referee) ; Fasurová, Naděžda (advisor)
A bachelor thesis was focused on fluorescence measurement by emission-excitation matrix method (EEM) and finding of the main fluorophores of soil humic and fulvic acids samples. The results of the position of the main peak of fulvic acid isolated from soil type Cambisol were compared and were similar to the position of Elliot soil FA standard II. For determining of secondary peaks of fulvic acids the synchronous method (3D SFS) at lower wavelengths was used.
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Characterization of microfludic chips
Vejrosta, Jakub ; Enev, Vojtěch (referee) ; Venerová, Tereza (advisor)
Nowadays, when we are moving to smaller and smaller scales, microfluidics is being used, which helps us to move from fluid devices to the dimensions of microfluidics, which may have a great application in the future. This work deals with the correct preparation and production of a microfluidic chip. Which consist of two parts. One part is the polydimethylsiloxane (PDMS) part of the chip, and the other is the coverslip. In this work, the production process of this chip is improved. The functionality of these chips and the time dependence of the age of the chip and the polydimethylsiloxane from which the chips are made were tested using fluorescence microscopy.
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Use of thin layer chromatography for fractionation and characterization of organic matter isolated from alginite
Solanský, Pavel ; Sovová, Šárka (referee) ; Enev, Vojtěch (advisor)
This diploma thesis is focused on the study of structure and physicochemical properties of organic fractions of humic substances, which were obtained by the method of thin-layer chromatography. Humic substances, which were used in this study, were isolated from a sample of Slovak alginite based on the procedure of the International Humic Substances Society (IHSS). The following analytical techniques were selected for the characterization of isolated humic substances: thermogravimetric and elemental analysis, molecular absorption spectroscopy (UV/Vis), Fourier transform infrared spectroscopy (FTIR) and steady-state fluorescence spectroscopy. Each organic fraction of humic substances were characterized by molecular absorption spectroscopy (UV/Vis) and steady-state fluorescence spectroscopy. Using steady-state fluorescence spectroscopy, humic substances were found to be composed of fluorophores of humic and non-humic (protein) character. Organic fractions corresponding to the humic fluorophores were characterized by a higher content of oxygen substituents on the aromatic nukleus, a higher degree of aromaticity and also a higher molecular weight. The aim of this diploma thesis was to design and optimize the process of organic matter fractionation for the purpose of detailed understanding of the structure and properties of humic substances, which were isolated from the sedimentary rock alginite. Based on this, the practical applicability of the thin layer chromatography method to significantly reduce the molecular heterogenity of the studied humic substances was evaluated.
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Study of decomposition of organic matter in soil using the method of "tea bags"
Lišková, Kateřina ; Sedláček, Petr (referee) ; Enev, Vojtěch (advisor)
The main aim of this master thesis was to introduce an optimal method which could be used for study of a decompostion of an organic matter in a soil. For this purpose was chosen based on a literaly review so-called tea bag method. With regard to the completeness of the work, a physico-chemical characterization of the soil environment and HS isolated from it was also performed. The degraded organic material was then further characterized using thermogravimetric analysis, elemental analysis, FT-IR spectroscopy, and the total content of water-extractable polyphenolic substances in the samples was also determined.
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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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Preparation and characterization of magnetic nanoparticles modified by surfactants
Solnička, Ondřej ; Másilko, Jiří (referee) ; Enev, Vojtěch (advisor)
Theme of this bachelors thesis was preparation and characterization of iron(II, III) oxide nanoparticles. Those nanoparticles were modified by surfactants. The surfactants which were used for modifiation were CTAB, TTAB and Septonex. Prepared and modified nanoparticles were taken for characterization by using methods of elemental analysis, thermogravimetry, dynamic light scattering and transmission electron microscopy (TEM). Results from each measurement shows that nanoparticles modified by CTAB had on average 85.9 nm in diameter, TTAB size was on average 108.3 nm and the last nanoparticles with Septonex had 246.6 nm in diameter on average. Prepared modified nanoparticles except the Septonex ones were stable enough to not aggregate. Septonex modified nanoparticles had greater tendenty to aggregate.
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Characterization of humic substances isolated from natural waste treated by vermicomposting process
Marková, Kamila ; Doskočil, Leoš (referee) ; Enev, Vojtěch (advisor)
This bachelor thesis deals with the study of humic substances isolated from vermicompost natural waste through spectrometric methods. The aim of this thesis is to study physicochemical properties of the humic substances through modern instrumental techniques such as molecular absorption spectroscopy in the ultraviolet and visible regions of electromagnetic radiation (UV/Vis), infrared spectroscopy with Fourier transformationn (FTIR), elemental analysis (EA), thermogravimetric analysis (TGA) and inductively coupled plasma emission (ICP OES). A research focused on basic information about the humic substances was developed in the theoretical part. A description about their distribution or structure was made and also their utilization in medicine, industry or ecology. Then, methods, which are used for study of the humic substances, were described as well. An essential part of the thesis was isolation of humic and fulvic acid from the vermicompost natural waste. Absorption coefficients were calculated from the measured UV/VIS spectrum which served to following characterization of HK and FK. FTIR spectrum was used to determination of functional groups of studied HL. An area of biogenic elements was specified using the elementary analysis. Total amount of studied trace elements was determined with with the help of ICP-OES.
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