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Characterization of extracellular enzymes and other metabolites of carotenogenic yeasts
Těšíková, Karolína ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
Lipases are enzymes catalyzing primarily the hydrolytic cleavage of triacylglycerol bonds. The production of lipolytic enzymes is known in many microorganisms, especially those who are able to utilize a fatty carbon substrate. Some genera of carotenogenic yeasts are characterized by this ability. Carotenogenic yeasts are characterized primarily by the formation of intracellular carotenoids, lipids and lipid-soluble substances. In addition to these metabolites, they may also produce some biosurfactants. This work deals with the production of extracellular lipolytic enzymes and biosurfactants by carotenogenic yeasts Rhodotorula glutinis, Cystofilobasidium macerans, Rhodotorula mucilaginosa and Sporidiobolus pararoseus cultivated mainly on animal waste fat at various C/N ratios (13, 25, 50, 100). Lipase activity was detected in all strains studied. Enzyme activities were measured by spectrophotometric method. Lipase induction has also been observed during cell growth, where several peaks of lipase activity have been reported, suggesting cell-associated lipase and lipase secreted into the environment. Lipase activities have also been found in cultures on glucose and glycerol carbon substrates. Further, the molecular characterization of lipolytic enzymes was performed using polyacrylamide gel electrophoresis. The formation of biosurfactants is to some extent formed by all strains. In particular, the biosurfactants of C. macerans and S. pararoseus yeast have emulsifying and solubilizing properties. Simultaneously with the production of lipase and biosurfactants, the production of characteristic high value added intracellular metabolites in S. pararoseus and R. mucilaginosa was evaluated too.
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Characterization of Rhodosporidium toruloides proteome using LC-MS/MS
Bruštík, David ; Szotkowski, Martin (referee) ; Zdráhal, Zbyněk (advisor)
Characterization of differentially regulated proteins is crucial for the identification of metabolic pathways and their understanding in connection with the creation of important products of a selected strain of yeast. This diploma thesis focuses on the proteome analysis of the Rhodosporidium toruloides cultivated under different conditions. The metabolism of these yeasts with the characteristics of important metabolites is described in the theoretical part. The next part of the thesis is focused on proteomic approaches and bottom-up proteomics from the sample preparation to mass spectrometry analysis. The experimental part deals with the cultivation of yeast at different C/N ratios, next the isolation and determination of proteins using the FASP method, which includes proteolytic cleavage by trypsin, LC-MS/MS analysis and the database search.
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Influence of stress and nutritional conditions on carotenogenic yeast and microalgal metabolism
Sikorová, Pavlína ; Byrtusová, Dana (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis describes the infuence of biological stress (co-cultivations) on the growth and metabolism of selected carotenogenic yeasts and microalgae. The metabolites of these microorganisms are chlorophyll and carotenoids, which are natural pigments and antioxidants. In theory, co-cultivation is a cultivation where microorganisms mutually support each other in the growth and production of metabolites. This would cause the increased production of previously mentioned chlorophyll and carotenoids. The theoretical part of the work is focused on the description of selected species of yeasts and microalgae and further discusses in more detail, the topics of stress factors depending on the growth and metabolism of microorganisms. The experimental part then deals with different types of cultivation and cocultivation and tries to optimize the production media and find the best symbiotic yeasts and microalgae. Furthemore the issue of different ratios of microorganisms in co-cultivations is also addressed here. The cultivated yeasts strains were Rhodotorula kratochvilovae, Rhodosporidium toruloides and Phaffia rhodozyma. And microalgae strains were Desmodesmus acutus, Desmodesmus quadricauda, Coccomyxa sp., Chlorella sorokiniana, Chlamydomonas reinhardtii and Scenedesmus obliquus. Cultivated cyanobacterium was Synechoccocus nidulans. The most successful co-cultivation experiment was the one with R. kratochvilovae and yeast Desm. quadricauda. This experiment was very succesful in all aspects.
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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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Production and characterization of immunologically active microbial beta-glucans by yeast.
Gerspitzerová, Nela ; Šimanský, Samuel (referee) ; Szotkowski, Martin (advisor)
The aim of this bachelor thesis is to study the effect of different cultivation conditions on the production of biomass, beta-glucans, lipids, carotenoids and other metabolites in selected strains of carotenogenic yeast. The thesis is divided into two main parts, a theoretical one and an experimental one. Theoretical part contains a characterisation of beta-glucans, their sources and uses, furthermore a description of the selected yeast genera and other metabolites they produce and finally possible analytical methods of these compounds. Experimental part describes the cultivation, analysis and obtained results. During the first experiment, the yeast strains were cultivated in production media with different C/N ratio, during the second experiment, the yeasts were cultivated either at room or at lower temperature with different content of phosphorus in the media. The selected strains of yeast were Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodosporidium toruloides, Cystofilobasidium macerans, Sporidiobolus metaroseus and Sporidiobolus pararoseus. Increasing C/N ratio generally had a positive effect on the production of lipids, while exhibiting the opposite effect on carotenoids, it did not have an unequivocal effect on the amount of beta-glucans. It was discovered that lowering the cultivation temperature had a significant negative effect on the production of all observed metabolites. Increased content of phosphorus in production media positively influenced the production of biomass and beta-glucans in most strains, on the other hand the highest amount of carotenoids was reached at lower concentration of phosphorus. The amount of phosphorus had almost no effect on the production and the profile of lipids. The highest amount of beta-glucans in biomass was discovered in C. macerans, their biggest production was reached by R. toruloides. The strain S. metaroseus was the most suitable for accumulation of lipids in its biomass. The highest amount of total carotenoids was reached by R. mucilaginosa.
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Production of carotenoids and other metabolites by yeast using selected food wastes.
Chujanov, Oleg ; Šimanský, Samuel (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis is focused on the issue of yeast cultivation with the usage of waste sub-strates from the food industry on a laboratory level. Carotenogenic yeasts are microorganisms capable of processing food waste substrates and use them as a source of organic nutrients for growth. This thesis consists of a practical as well as theoretical part. The experimental section focuses on the description of different strains of yeasts, metabolites and types of waste sub-strates. The two main substrates chosen for this research were chicken fat and chicken fe-athers. The yeast types used in this thesis were Rhodotorula kratochvilovae (CCY 20-2-26), Rhodosporidium toruloides (CCY 62-2-4), Cystofilobasidium macerans (CCY 10-1-2), Spo-ridiobolus pararoseus (CCY 19-9-6), Rhodotorula mucilaginosa (CCY 20-9-7), Rhodotorula mucilaginosa (CCY 19-4-25) and Sporidiobolus metaroseus (CCY 19-6-20). These strains reported high production values of the metabolites researched in this thesis. The strains Rho-dosporidium toruloides (CCY 62-2-4) and Sporidiobolus metaroseus (CCY 19-6-20) were chosen for the production of metabolites from waste substrates, for those reported the most promising results.
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A comparison of metabolic activity and use of microalgae and pigment-formed yeasts
Szotkowski, Martin ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
Carotenoids are natural pigments occurring in plants and many microorganisms, such as algae, yeast and bacteria. They represent the most common group of antioxidants with significant biological effect. Lipids are an essential component of all living organisms. They are the source and the reservoir of energy for organisms, which can also be used in the petrochemical industry for the production of biofuels. This thesis deals with the production properties of carotenoids and lipids by selected strains of yeasts, algae, and canobacteria, in order to apply the acquired knowledge and find cheap suitable alternatives for microbial biotechnological production of these metabolites using waste substrates. Carotenoids, coenzyme Q, and ergosterol in cells were determined by liquid chromatography. The lipid content and fatty acid profile was analyzed by gas chromatography. Cell morphology and localization of selected metabolites were analyzed by fluorescence lifetime imaging microscopy. In this work yeast genera Sporobolomyces, Cystofilobasidium and Rhodotorula were used. As the representatives of the algae and cyanobacteria strains of Desmodesmus, Cyanothece, Chlamydomonas, Synechoccocus and Chlorella strains were studied. In the overall comparison, the yeast strains were more productive than algae and cyanobacteria. The highest carotenoid production was found in S. pararoseus, C. capitatum and R. mucilaginosa cells, while the highest lipid yield was observed in strains of C. infirmominiatum and S. metaroseus.
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Cocultivation of yeasts and microalgae to produce enriched biomass
Bradáčová, Lenka ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The diploma thesis is focused on the influence of biological stress formed by co-cultivation of heterotrophic (yeasts) and autotrophic (microalgae and cyanobacteria) organisms on the production of enriched biomass. The monitored groups of substances include carotenoids (-carotene, lutein, lycopene, astaxanthin, torularhodin), chlorophylls A and B, ergosterol and ubiquinone. Further, production of lipids was analyzed in the terms of fatty acid profile and lipid content in biomass. In the first part of the work, the yeast biomass production was investigated using different nitrogen sources. Glycerol was used as a carbon source in all parts of the work. Subsequently, the co-cultivation of yeasts with microalgae and cyanobacteria took place in a multicultivator with gradual increase of selected macroelements – nitrogen, magnesium and phosphorus. The last part of the work was focused on the co-cultivation of yeasts and microalgae in a laboratory fermenter. The best effect on the production of total biomass was the increased magnesium content and high nitrogen content in the basic medium. The best concentrations of carotenoids were achieved due to the double nitrogen and phosphorus content together. Chlorophyll production was significantly lower compared to carotenoids.
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Characterization of biological effects of extracts obtained from yeasts and autotrophic microorganisms
Špačková, Dominika ; Bendová, Agáta (referee) ; Szotkowski, Martin (advisor)
Carotenogenic yeasts and microscopic autotrophic microorganisms are increasingly being studied in the biotechnology industry because of their ability to produce a variety of substances with a wide range of utilization. These substances mainly include lipids and carotenoids, which are used in the food and pharmaceutical industries. The aim of this bachelor thesis is to determine the way these microorganisms behave in the presence of stress factors, and which of them would have a positive effect on the production of metabolites that can be further used in various industries. The theoretical part is dedicated to a characterization of substances produced by carotenogenic yeasts, cyanobacteria and microalgae, which will be further analyzed in the experimental part. The metabolites mainly include carotenoids, which are a wide group of naturally occurring pigments that possess, among other things, antioxidant and provitamin activity. No less important substances produced by these organisms are lipids, which are part of cell membranes, they also serve as a reservoir of energy, and the substances contained in lipids are part of the precursors of many hormones. Yeast cell walls also contain -glucans, which have many beneficial effects on immunity and human health. Due to this fact, interest in their research have been rising recently. The analytical methods used to determine these substances are also mentioned, as well as stress factors and their effects on the cells of the mentioned organisms. The experimental part deals with the exposure of these microorganisms to oxidative stress and stress caused by heavy metals. The aim was to analyze which concentrations of these stress factors are lethal to microorganisms, and which of them are optimal for increase of the production of the above-mentioned substances. Of the yeast strains, the strains Sporidiobolus pararoseus and Rhodotorula mucilaginosa achieved increased production of metabolites due to selenate ions. In contrast, cobalt had a beneficial effect only on the accumulation of lipids and -glucans in cells. The positive effect of selenium was also observed in the strain Chlorella minutissima and Arthrospira maxima, however, the most remarkable changes in the production of metabolites were achieved by the addition of the optimal concentration of the solution inducing oxidative stress.
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Comparison of growth and productivity of autotrophic algae and red yeasts
Kouřilová, Xenie ; Rapta, Marek (referee) ; Márová, Ivana (advisor)
The submitted bachelor thesis focuses on the comparison of growth and productivity of red yeasts Rhodotorula aurantiaca and Xanthophyllomyces dendrorhous and autotrophic algae Chlamydomonas reinhardtii and Chlorella vulgaris under different physical conditions. The theoretical part contains the characteristics of selected micro-organisms, stress factors caused by temperature, radiation, combination of aeration and radiation and osmotic stress. Furthermore, knowledge on metabolites and the possibilities of their analysis are mentioned. Physical stresses, namely 2%, 3%, 4% and 5% solution of sodium chloride, intense radiation, absence of lighting, 8°C and 30°C, were applied to selected strains in cultivation. Under these conditions, changes in production of carotenoids, fatty acids, ergosterol, ubiquinone and proteins were being observed. A significant inhibitive effect on all the above mentioned micro-organisms was brought about by the use of 5% solution NaCl. The highest production of metabolites with X. dendrorhous was discovered when 4% solution of NaCl was used. R. aurantiaca demonstrated higher substance utilization at 30°C. For the selected algae strains were the used stresses lethal. Within the cultivation process, the possibilities of co-cultivation of yeasts and algae were also tested, which will be the subject of further studies.
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