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Cultivation of carotenogenic yeasts in the presence of biological stress induced by selected strains of autotrophic microalgae
Sikorová, Pavlína ; Byrtusová, Dana (referee) ; Szotkowski, Martin (advisor)
Presented Master’s thesis was focused on biological and nutrient stress in cultivations of green microalgae and carotenogenic yeasts. The focus of interest was production of biomass in different types of cultivation media, production of metabolites (mainly carotenoids and chlorophylls) and also production of lipids and lipophilic substances. All types of aplied stress was used to lead to an increased production of biomass and metabolites. The theoretical part deals with introduction of individual genera of carotenogenic yeast and green microalgae. The types of cultivation stress were also described. In addition, the analytic methods for qualitative and quantitative analysis are also introduced. The experimental part was focused on the study of biomass, carotenoids, chlorophylls, coenzym Q, sterols and lipids production. Biological stress was induced by cocultivation of microalgae and yeasts together.Nutrient stress meant adding macroelements or waste oils to the medium. Yeasts of interest were Rhodosporidium toruloides, Rhodotorula kratochvilovae and Sporobolomyces pararoseus. Microalgae was represented by Desmodesmus acutus, Desmodesmus armatus, Scenedesmus obliquus, Desmodesmus velitaris, Desmodesmus communis, Coccomyxa sp. Chlamydomonas reinhardtii and Chlorella minutissima. Generally the most successful multicultivator experiment was cocultivation with yeast R. kratochvilovae. In the case of cultivation on waste oils, the best producers of biomass and metabolites were co-cultivation experiments with the yeast R. toruloides. In microalgal experiments, it was found that nutritional stress in the form of glycerol added to the medium had an inhibitory effect on the growth and metabolism of microalgae.
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Analysis of nicotin content in some products
Pražáková, Jana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the determination of nicotine in different products. The theoretical part summarizes review on nicotine, smoking and opportunities how to quit. In the practical part a method for the determination of nicotine by HPLC / PDA was optimized. As the most suitable stationary phase was selected a Kinetex 5u C18 100A 150 x 4.6 mm column, as the optimal mobile phase was chosen a pure methanol with a flow rate of 1 ml min-1 and a temperature of 25 °C. For the analysis of nicotine were chosen: 18 kinds of cartridges for electronic cigarettes, two kinds of nicotine gum, nicotine spray, nicotine pastilles, nicotine orodispersible film and ten species of classic cigarettes. For each type of product the most appropriate method for extracting nicotine and its subsequent analysis by HPLC / PDA was found. For tobacco 24 hour extraction in methanol and 10s ultrasound was selected. The nicotine spray and electronic cigarette refills without flavours were only diluted with methanol. Flavoured refills were first diluted by sodium hydroxide and then with methanol. For chewing gums, pastilles and nicotine film extraction with 5% sodium hydroxide was chosen. In this study also new experimental nicotine product was designed. Nicotine has been encapsulated in alginate-starch material to form small gel particles. As the most suitable medium for storage the water medium was determined.
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Use of carotenogenic yeasts to production of lipid soluble metabolites
Mariničová, Veronika ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
Carotenoids are fat-soluble chemical compounds that occur as natural pigments in many plants and protect them from sunlight. Lipids are also essential lipophilic substances and they are part of biomembranes. Their main function is primarily to serve as a power supply for the cell, protective function and thermal protection against adverse environmental influences. This bachelor thesis deals with cultivation of selected carotenoid yeast genes, subsequent isolation of carotenoids and other lipid substances, which can be used as a source of potentially beneficial substances for the pharmaceutical or cosmetic industry. The theoretical part deals with the description of carotenoid yeasts, chemical composition and biosynthesis of the metabolites produced, and description of the methods used for their determination. The experimental part is focused mainly on the production of lipid substances by various strains of yeasts using cheap waste substrates and the application of exogenous stress (nutritional stress) to the biotechnological overproduction of selected metabolites using the modification of the production medium. The content of carotenoids, ergosterol and coenzyme Q was analyzed by high performance liquid chromatography with a PDA detector. The lipids accumulated in yeast cells were determined by gas chromatograph with a FID detector. In this work the strains of Sporobolomyces pararoseus, Sporobolomyces metaroseus, Rhodotorula glutinis and Cystofilobasidium infirmominiatum were studied. As waste substrates and carbon sources were used glycerol, which is produced as a waste product in the production of biofuels and whey as an unusable product in dairy technology. The best production on waste substrates was observed in the strains Rhodotorula glutinis and Cystofilobasidium infirmominiatum.
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Co-cultivation of carotenogenic yeasts and cyanobacteria on selected food wastes
Kodajek, Matěj ; Márová, Ivana (referee) ; Szotkowski, Martin (advisor)
Cyanobacteria and yeast are microorganisms, that are cultivated in the biotechnological industry to produce biomass. Biomass contains several organic substances, that are used in the food, pharmaceutical or energy industries. This thesis deals with the joint cultivation (co-cultivation) of cyanobacteria and yeast on food waste. The cyanobacteria Synechococcus nidulans, Synechococcus bigranulatus, Anabaena torulosa and Spirulina were used for co-cultivation with yeast Rhodotorula kratochvilovae and Rhodotorula toruloides. Cultivation media included following food wastes: coffee oil, hemp oil, frying oil, molasses, molasses hydrolyzate, whey, whey hydrolyzate and waste glycerol. In the theoretical part is a comprehensive literature research focused on cyanobacteria, yeasts, and their specific genera, monitored metabolites, co-cultivation, food waste and analytical methods. The experimental part contains a list of used chemicals, devices, aids, strains, and descriptions of procedures for the cultivation of microorganisms, isolation, and analysis of biomass. The aim of the thesis was to do screening cultivations, to find suitable strains of microorganisms for symbiotic co-cultivation and to find a suitable food waste. Following co-cultivation in a semi-operational laboratory bioreactor. Analysis the obtained biomass using gas and liquid chromatography and evaluation the results.
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Analysis of authenticity of some food products with fruit component by molecular and instrumental techniques
Pecháček, Michal ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This master’s thesis was focused on determining the authenticity of some food products with fruit component by molecular and instrumental techniques. The thesis was divided into theoretical and practical part. The theoretical part of the work was focused on food adulteration, methods of determining the food authenticity, analysed types or technologies of production of individual food products, which were analysed in this work. This part was also focused on molecular and instrumental techniques, which are currently used to determine the food authenticity. The practical part of the thesis was divided into molecular and instrumental part. Within the molecular part, DNA isolation was performed using the EliGene Plant DNA Isolation kit. Inhibitors such as polysaccharides seemed to be the biggest problem during the DNA isolation. Therefore, an isolation method using pectinase incubation was performed. The isolated DNA was subsequently subjected to PCR and the resulting PCR products were analysed by a melting curve analysis. This method was used for ITS2 primers, which were used for the plant DNA detection. In the case of species-specific primers BAS1 and Pa3LTP that were used for the peach and apricot detection, high resolution melting (HRM) analysis was performed. During HRM, the focus was on the melting temperature of the specific PCR products. The melting temperature of the BAS1 specific product was set at 78,4 °C and at 86,43 °C for the specific product of Pa3LTP primers. Finally, specific PCR products were subjected to agarose gel electrophoresis. In the case of ITS2 primers, which served to verify the amplifiability of plant DNA, a band of 500 bp was detected. In the case of species-specific primers BAS1 and Pa3LTP, bands from 100 to 150 bp were detected. In the instrumental part of this thesis the HPLC/PDA analysis of the phenolic compounds was performed. The most suitable procedure for the analysis of phenolic substances was the purification procedure using ethanol without further concentration At the end of the work, the individual methods were compared with each other. At the same time, the influence of the matrix on the overall determination of food authenticity was monitored. While molecular techniques could be used to determine the food authenticity by determining the presence of specific DNA, instrumental techniques would be more suitable for detecting food adulteration and detecting specific substances.
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Cultivation of carotenogenic yeasts in the presence of biological stress induced by selected strains of autotrophic microalgae
Sikorová, Pavlína ; Byrtusová, Dana (referee) ; Szotkowski, Martin (advisor)
Presented Master’s thesis was focused on biological and nutrient stress in cultivations of green microalgae and carotenogenic yeasts. The focus of interest was production of biomass in different types of cultivation media, production of metabolites (mainly carotenoids and chlorophylls) and also production of lipids and lipophilic substances. All types of aplied stress was used to lead to an increased production of biomass and metabolites. The theoretical part deals with introduction of individual genera of carotenogenic yeast and green microalgae. The types of cultivation stress were also described. In addition, the analytic methods for qualitative and quantitative analysis are also introduced. The experimental part was focused on the study of biomass, carotenoids, chlorophylls, coenzym Q, sterols and lipids production. Biological stress was induced by cocultivation of microalgae and yeasts together.Nutrient stress meant adding macroelements or waste oils to the medium. Yeasts of interest were Rhodosporidium toruloides, Rhodotorula kratochvilovae and Sporobolomyces pararoseus. Microalgae was represented by Desmodesmus acutus, Desmodesmus armatus, Scenedesmus obliquus, Desmodesmus velitaris, Desmodesmus communis, Coccomyxa sp. Chlamydomonas reinhardtii and Chlorella minutissima. Generally the most successful multicultivator experiment was cocultivation with yeast R. kratochvilovae. In the case of cultivation on waste oils, the best producers of biomass and metabolites were co-cultivation experiments with the yeast R. toruloides. In microalgal experiments, it was found that nutritional stress in the form of glycerol added to the medium had an inhibitory effect on the growth and metabolism of microalgae.
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Metabolic adaptation of selected microalgal strains on various nitrogen sources
Kodajek, Matěj ; Šimanský, Samuel (referee) ; Szotkowski, Martin (advisor)
Microalgae and cyanobacteria are photosynthetic organisms that, together with other microorganisms (yeast, bacteria), are used in industry, because they produce a wide range of interesting organic substances. This thesis deals with the metabolic adaptation of microalgae Scenedesmus obliquus, Scenedesmus acutus, Scenedesmus dimorphus, Chlamydomonas reinhardtii, Coccomyxa sp. and cyanobacteria Synechococcus nidulans, Arthrospira maxima, Limnospira maxima, Anabaena torulosa, which were cultivated on various nitrogen sources. These strains are descibed in the theoretical part including the metabolites and their applications in industry. The experimental part describes all chemicals, aids, devices and methods used for cutivation and analysis of microorganisms. The aim of the study was to compare and find out which nitrogen source in the BBM and SPIRULINA medium is the most suitable for a particular strain in terms of production of total biomass and composition of individual metabolites. Sodium nitrate, ammonium sulfate, glycine, urea and whey protein were used as nitrogen sources. The content and composition of lipids in the biomass was determined by gas chromatography. The content of ubiquinone, carotenoids and chlorophylls was determined by liquid chromatography.
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Production of pigments and lipid substances by microorganisms on waste substrates of the food industry
Hladká, Dagmar ; Němcová, Andrea (referee) ; Szotkowski, Martin (advisor)
The presented study is focused on production of carotenoids, lipids and other substances by carotenogenic yeasts and autotrophic algae. Studied strains were cultivated in media with different composition, including waste substrates from food industry. Studied strains were cultivated under stress conditions to enhance the production of desired metabolites. The theoretical part deals with the information about yeasts and algae, with the information about monitored metabolites such as lipids, carotenoids, ergosterol, ubichinon or chlorophyll. Furthermore, the theoretical part deals with possible methods of metabolite analysis. The experimental part deals with the description of cultivation of yeats and algae. Also experimental part is focused on the description of individual methods. The result part deals with comparition of production of biomass, metabolits and lipids. The selected strains of yeast were Sporidiobolus pararoseus, Sporidiobolus metaroseus, Sporobolomyces roceus, Phaffia rhodozyma and Dioszegia hungarica. The selected strains of algae were Desmodesmus acutus, Desmodesmus quadricauda, Scenedesmus dimorphus and Chlamydomonas reinhardtii. We were optimized conditions for metabolites and lipids production. Optimal medium, which contained coffee hydrolyzate was with carbon to nitrogen ratio C/N 25. Nitrogen in higher concentration had negative effect on production of lipids. The most suitable strain for effective use of nitrogen in different concentrations was Chlamydomonas reinhardtii. The most suitable strain of yeast for effective use of coffee hydrolyzate was Sporidiobolus metaroseus.
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Optimization of extraction of pigments from yeast and algae cells
Šimanský, Samuel ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The presented diploma thesis deals with the extraction and storage stability of lipophilic pigments produced by selected strains of yeasts and algae. In this thesis, there was studied the influence of the selected solvents on the efficiency of extraction, as well as the effect of ambient temperature on the stability of the pigments during storage. The work is divided into two main parts, theoretical and practical part. In the theoretical part knowledge about algae, yeasts and their cultivation parameters is summarized. Furthermore, some information regarding the selected lipid metabolites, their properties and possibilities of application in various branches of industry is mentioned. The practical part deals with the preparation of extracts and stability tests. Extracts were prepared from selected biomass samples in solvents suitable for applications in food industry or cosmetics (ethanol and hexane). Subsequently, the long-term stability tests lasting 4 months and short-term stability tests lasting a total of 28 days were performed on these extracts. The pigments were determined by HPLC and spectrophotometrically, the fatty acid content was determined by GC. For the extraction of pigments from biomass, in the most cases ethanol appears to be the optimal solvent. However, for lipid extraction from biomass, hexane appears to be the optimal solvent for a significant number of samples. In most samples, storage in the freezer showed the most favourable effect on pigment stability, but some samples showed comparable stability even when stored in the refrigerator.
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Production of selected metabolites by yeasts and algae cultivated under stress conditions
Mariničová, Veronika ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
The presented work was focused on the comparison between the production of selected metabolites by carotenogenic yeasts and microalgae cultivated under conditions of external stress. The main metabolites of interest were carotenoids, further lipophilic substances and lipids. Biotechnological overproduction of these metabolites could serve as a source of potentially beneficial substances not only for the pharmaceutical, cosmetic and food industries, but also for the production of third generation biofuels. Recently, there has been a growing interest in biofuels primarily from microalgae, which have a high potential in biofuel production and seem to be a promising source. The theoretical part deals with the description of individual genera of carotenogenic yeasts, microalgae, cyanobacteria, chemical composition of produced metabolites and brief biosynthesis. In addition, individual methods for analyzing the production of the metabolites of interest were described. The experimental part is focused on the comparison of production of carotenoids, coenzyme Q, ergosterols (phytosterols) and lipids by yeasts, microalgae and cyanobacteria. As a source of external stress, temperature, salt and light stress were chosen. The strains of Rhodotorula glutinis, Rhodotorula mucilaginosa, Sporidiobolus pararoseus and Cystofilobasidium macerans were studied from the yeast strains. Microalgae and cyanobacteria were Scenedesmus obliqus, Scenedesmus dimorphus, Chlorella sorokiniana, Chlorella saccharophila, Botryococcus brauni, Synechococcus nidulans and Arthrospira maxima. The yeast and algal strains were optimized for growth, carotenoid and lipid production. Applied salt stress showed a significant liquidation effect on algal and cyanobacterial strains. The thesis also monitored the biological stress, so-called co-cultivation of microalgae and yeasts. Further experiments will be the subject of future work.
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