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Study of the production of polyhydroxyalkanoates using selected thermophilic representatives of the genus Aneurinibacillus
Řeháková, Veronika ; Kovalčík, Adriána (referee) ; Pernicová, Iva (advisor)
The subject of the diploma thesis is the production of PHA copolymers by thermophilic Gram-positive bacteria of the genus Aneurinibacillus. The theoretical part is devoted to the general characterization of PHA materials, their properties, use, and also to the production of PHA polymers by these bacteria. The experimental part deals with the production of PHA copolymers using selected thermophilic members of the genus Aneurinibacillus. Firstly, the specific enzyme activity of PHA synthases was determined, and then the production of PHA copolymers was screened using selected lactones (-valerolactone, -hexalactone, -valerolactone) and diols (1,6-hexanediol, 2,3- butanediol and 1,4-butanediol). These experiments were performed with six thermophilic producers by the members of the genus Aneurinibacillus, which were isolated from compost and activated sludge. Tested microbial strains have demonstrated the ability to integrate interesting monomers into the PHA structure, including 4-hydroxyvalerate (represented up to 69.3% of the total PHA), 5-hydroxyvalerate (up to 47.1%), or 4-hydroxyhexanoate (up to 31.9%). Subsequently, a closer characterization of the obtained PHA (which were gained by the production of the best producers) using advanced methods (DSC, SEC-MALS, FT-IR) was performed. Finally, the screening of the PHA copolymers production was performed in laboratory bioreactors.
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Microbial synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)]
Dugová, Hana ; Zemanová, Jana (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis studied the ability of Cupriavidus malaysiensis, Delftia acidovorans and Azohydromonas lata to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)], by using -butyrolactone and 1,4-butanediol as carbon substrates. The objective of this work was the production and characterisation of isolated polyhydroxyalkanoates (PHA). The theoretical part deals with the basic description and classification of polyhydroxyalkanoates. Next, the biosyntheses of the most investigated PHAs were described. The practical section of the work discusses and presents the output of the cultivation of five bacterial strains selected for the production of [P(3HB-co-4HB)], namely, Cupriavidus malaysiensis (DSM 19379), Delftia acidovorans (DSM 39), Delftia acidovorans (CCM 2410), Delftia acidovorans (CCM 283) and Azohydromonas lata (CCM 4448). The effect of the modified cultivation conditions for each of the used bacteria on the PHA production yields was discussed. The produced biomass after the cultivation was characterised spectrophotometrically, gravimetrically and by gas chromatography. Polymers were isolated from the biomass by the extraction in chloroform. The isolated polymers were characterised from the viewpoint of chemical composition, molecular weight and thermal properties by using Attenuated total reflection infrared spectroscopy, Size exclusion chromatography, Differential scanning calorimetry and Thermogravimetric analysis.
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Preparation and characterization of packaging films based on beta-keratin
Měšťánková, Zuzana ; Mikulíková, Renata (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis deals with keratin isolation from poultry feather waste and preparation of keratin-polymers composite films utilizing isolated keratin. The main goal was to isolate keratin from poultry feathers by hydrolysis and compare yield of diverse hydrolysis types. The most efficient was hydrolysis using 0.5% sodium hydroxide with yield 480 g of keratin per 1 kg of chicken feathers. Structure, morphology and thermal properties of isolated keratin was analyzed. Comparison with protein molecular weight standard it was revealed that hydrolysis cleaved keratin to macromolecules smaller than 15 kDa. Scan electron microscopy showed that size of lyophilized keratin particles was in the order tens of µm and had an irregular shape. Further, composite films based on isolated -keratin and other polymers were prepared. Thermal analysis showed, the addition of keratin significantly reduced the thermal stability of composite films. Analyses revealed that Polyvinyl alcohol composite films reached the best mechanical properties. For example, an addition of 0.25 g of keratin per 1 g of polyvinyl alcohol increased the tensile elongation by up to 68.9%. Moreover, the film based on chitosan and keratin had antibacterial properties. The results demonstrate that it is able to efficiently isolate keratin from poultry feather waste by alkaline hydrolysis and isolated keratin can be utilized to prepare potentially applicable keratin-polymers composite films.
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Production of PHB-HV from grape pomace by employing a pure culture of Cupriavidus necator
Jakešová, Michaela ; Kučera, Dan (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deal with the production of PHB-HV copolymer employing a pure culture of Cupriavidus necator H16. First, Erlenmeyer flask´s cultivations using pure fructose as a source of carbon were performed. The mode of addition of valeric acid for the synthesis of PHB-HV was studied. Furthermore, the effect of stress factor (ethanol) on PHB-HV synthesis by this microorganism was investigated. The method obtained by optimization in Erlenmeyer flasks was applied to the synthesis of the PHB-HV copolymer in the bioreactor using fructose as a source of carbon, too. Furthermore, after enzymatic hydrolysis, a sugar extract from grape pomace was obtained as a source of carbon for the synthesis of PHB-HV. The fermentation in Erlenmeyer flasks and the bioreactor using an extract of grape pomace was carried out. Using this alternative carbon source 3.51 g/l PHB-HV copolymer was produced in Erlenmeyer flask, 5.06 g/l in bioreactor respectively. In the first case, the proportion of 3-hydroxyvalerate in the copolymer was 23.78 mol. % and in the second one, 9.39 mol. %.
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Study of 3D bacterial cellulose production using banana peels as a carbon source
Netopilík, Tibor ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the study of 3D bacterial cellulose production using Komagataeibacter xylinus using banana peels as a carbon source. The theoretical part deals with the comparison of the properties of bacterial cellulose and plant cellulose, different methods of biotechnological production of bacterial cellulose and its use. The aim of the bachelor thesis was to find out whether it is possible to use banana peel as a carbon source for biotechnological production of bacterial cellulose. Banana peels are waste lignocellulosic material produced, for example, in the production of snacks or fruit or dairy beverages in the food industry. HPLC analysis showed that 1 l of hydrolyzate after enzymatic hydrolysis of 100 g of dry banana peels per 1 l of water contained 8.86 g of glucose and 10.46 g of fructose. The hydrolyzate was used as a carbon source for static and dynamic cultivation of Komagataeibacter xylinus. The yields of bacterial cellulose produced by using banana peels or glucose were compared. The use of banana peels hydrolyzate increased the yields of bacterial cellulose about 170 % in the case of static cultivation and about 220 % in the case of dynamic cultivation. Scanning electron micrographs of bacterial cellulose confirmed the morphological differences between bacterial cellulose produced statically and dynamically. Moreover, the morphology of bacterial cellulose was influenced by the kind of used carbon source.
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Use of technical hemp in acne preparations
Žáčková, Kristýna ; Kovalčík, Adriána (referee) ; Němcová, Andrea (advisor)
Hemp belongs among important plants with a long industrial tradition. However, its first use was originally for healing. In medicine, it is mainly applied thanks to the content of many biologically active substances, such as cannabinoids. This thesis is concerned with the use of hemp in acne preparations. The theory focuses mainly on the characterization of hemp and its active substances. The other chapters deals with cosmetic preparations and acne itself, and Propionibacterium acnes as well, which is involved. Within the experimental part, the extracts of two hemp varieties (Finola and Bialobrzeskie) were characterized in terms of antioxidant and cannabinoid presence. The cosmetic preparations with hemp extract content were prepared including a complete cleansing set for acne skin, concurrently commercial products of this sort were purchased. In cosmetic preparations and the extracts their inhibitory effect against Propionibacterium acnes was studied as well. Also Micrococcus luteus, Escherichia coli and Candida glabrata were tested. The results show that dried hemp flower extracts contain the greatest amount of active substances, so they have a higher antioxidant and antimicrobial effect. The presence of individual cannabinoids differed mainly depending on the variety. All hemp extracts had effect against gram-positive bacteria, in cosmetic preparations it was lower. Nevertheless hemp antibacterial effect has been proven.
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Determination of the effect of dog rose (Rosa canina L.) on the oxidative stability in fresh juices
Borová, Šárka ; Mikulíková, Renata (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis aimed to determine the content of bioactive substances and antioxidant activity in ethanolic extracts of rose hips fruits (Rosa Canina L.). This work verified the possibility of using the rosehip ethanolic extract as an antioxidant to inhibit the oxidation of fresh pear juice. The ethanolic extracts were prepared by extracting rose hips fruits in various ratios of fruits to the volume of 40% ethanol for 7-28 days. After determining the total content of carbohydrates, polyphenols, flavonoids and vitamin C, it was found that, in principle, the range of the defined substances increases with the ratio of rosehip fruit to the volume of ethanol solution as well as with the extraction time. Ethanol rosehip extracts have also been shown to possess significant-high antioxidant activity. The stabilizing effect of rosehip fruit extract against the oxidation of fresh pear juice was determined spectrophotometrically. It was found that rosehip fruit extracts could inhibit the oxidation of fresh pear juice and have the potential as a natural stabilizer of fresh fruit juices.
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Smart food polymer layers
Valíková, Michaela ; Kovalčík, Adriána (referee) ; Veselá, Mária (advisor)
Smart packaging represents a group of packaging with great potential when used in the food industry. Active packaging can have a positive effect on the condition of stored food and smart packaging can inform consumers about the condition of stored food. In the theoretical part, a search was made on the topic of packaging in the food industry, with emphasis on active and intelligent packaging, including the legislative framework. The substances from which smart packaging is made have been described. In the experimental part, partially water¬insoluble polymer layers were prepared from gelatin, glycerol, crosslinking agent and anthocyanin extract. Glutaraldehyde or tannic acid was used as a crosslinking agent. Anthocyanins were obtained by extraction from red cabbage. The total concentration of anthocyanins in the extract was determined by the difference method. For prepared layers, their water solubility was measured and the colour response to the pH environment was evaluated spectrophotometrically. The stability of the layers over time was also monitored. It was found that the prepared layers are able respond to changes in pH.
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Biotechnological production of PHA employing Cupriavidus malaysiensis
Drábková, Kateřina ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
The subject of this diploma thesis is the study of the production of polyhydroxyalkanoates (PHA) employing Cupriavidus malaysiensis. The aim of this thesis is to obtain and characterize the polymer with the most advantageous properties. The theoretical part deals with polyhydroxyalkanoates and their biotechnological production. Then selected microorganisms producing 3-hydroxybutyrate and 4 hydroxybutyrate, P(3HB co 4HB), are described. After that, the theoretical part deals with the production of PHA employing Cupriavidus malaysiensis. The bacterial strains of Cupriavidus malaysiensis DSM 19416, 19379 and 25816 were used in the experimental part. The strain Cupriavidus necator H16 was used as a control strain. First, the ability to utilize various carbon sources by the given bacterial strains was determined. Then, the aim was to select the optimal carbon source, a precursor for the synthesis of a polymer with high content of 4-hydroxybutyrate, and to determine the ideal culturing conditions for the growth of the microorganisms, which have been studied, and the production of P(3HB co-4HB). A two-stage culture and bioreactor culture was also performed to produce the desired copolymer. Furthermore, the production of terpolymer of 3-hydroxybutyrate, 4 hydroxybutyrate and 3-hydroxyvalerate, P(3HB co 4HB co 3HV), was studied by single stage and two-stage cultivation. In the last part, the produced polymers were characterized by gas chromatography with flame ionization detector (GC-FID), size exclusion chromatography with multi-angle static light scattering (SEC-MALS), differential scanning calorimetry (DSC) and thermogravimetry.
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Monitoring the influence of stress factors on yeasts of the genus Metschnikowia using molecular techniques
Kuljovská, Tereza ; Kovalčík, Adriána (referee) ; Němcová, Andrea (advisor)
Molecular techniques are used widely nowadays, especially in the identification of various species of microorganisms, and provide reliable and accurate results in a relatively short period of time. This work deals with the characterization of Metschnikowia yeasts exposed to various stress factors and the monitoring of their genomic DNA by PCR-DGGE and PFGE methods. The work was focused on the analysis of ribosomal rDNA, specifically the ITS1 and 5,8-ITS2 regions and genes encoding the domains D1/D2, that are part of the large 26S rDNA ribosomal subunit, which are commonly used in the characterization of fungal eukaryotic communities. Three types of stress factors were selected for the experiments: osmotic stress (NaCl environment), oxidative stress (addition of H2O2 to the medium) and nutritional stress (addition of hemp flour / leaves and flowers as a carbon source). The analysis was performed for particular strains Metschnikowia andauensis, Metschnikowia pulcherrima, M. chrysoperlae, M. shanxiensis, M. sinensis and M. zizyphicola. The results showed that addition of the NaCl, H2O2 and hemp components at higher concentrations to the production media does not disrupt the ribosomal DNA when detected by PCR-DGGE. Mutations have not been observed by comparing these strains with yeast that was cultivated under optimal conditions. Despite stress factors, PFGE analysis of karyotypes showed that DNA of some yeast species does not prove any damage and remain intact. Remaining strains proved certain degree of damage, and bands were not detected on the gel for these strains. In the given circumstances, it can be stated that the high adaptability of these species to a stress environment makes them promising biotechnology producers. These yeasts have great potential for usage in agriculture as a tool for biocontrolling of fruit or vines.
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