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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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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Production of pigments by yeasts
Gonová, Dominika ; Hlaváček, Viliam (referee) ; Němcová, Andrea (advisor)
Carotenoids are naturally occurring pigments synthesized by yeast, bacteria, filamentous fungi and plants. In recent years, the interest in the study of these pigments and their microbiological production is increasing mainly due to significant biological effects attributed to carotenoids. This work is conceived as comparative study of six carotenogenic yeasts: two strains of species Rhodotorula glutinis, Rhodotorula mucilaginosa, Sporobolomyces roseus, Sporobolomyces metaroseus a Sporobolomyces pararoseus. Their cultivation was carried out in various media where the oxidative stress was applied in the form of hydrogen peroxide and also waste material – egg pasta – was used as a nutrition source. All studied strains were able to use waste substrate as a source of nutrients, in case of the genus Sporobolomyces increasing production of metabolites was observed. Under the oxidative stress, the majority of the studied yeast showed overproduction of carotenoids and accompanying lipid substances. Sporobolomyces roseus CCY 19-6-4 was characterized by the highest production of carotenoids (1923.78 µg/g -carotene) at the egg pasta media, but on the other hand, it showed a significant decline in biomass. As potentially the best producer of biomass (9.85 g/l) and carotenoids (780.24 µg/g of -carotene), the yeast Rhodotorula glutinis CCY 20-2-26 was observed. Pulcherrimin, synthesized mainly by yeast but also by some spore-forming bacteria, also belongs among natural pigments. Recently, its antagonistic effect that was shown against several microorganisms has been intensively studied, as it seems to be very relevant in regard to biological control. The second part of the work deals with the regulation of pulcherrimin production and its antimicrobial activity. As the pigment-producing yeast strains were used Metschnikowia pulcherrima 145, Metschnikowia pulcherrima 147, Mestchnikowia pulcherrima 149, Metschnikowia andauensis 129, which were cultured on various media containing different concentrations of metal ions and hydrogen peroxide as exogenous stress. In the study of pulcherrimin antagonistic effect, gram-positive bacteria Lactobacillus sakei, gram-negative bacteria Escherichia coli, yeast Candida glabrata and filamentous fungi Phanrochaete chrysosporium were used as tested microorganisms. All studied yeasts were able to produce pulcherrimin, and therefore to show antagonist effect, only in media supplemented with iron, while the color intensity was proportional to the concentration of iron. Antimicrobial activity of pulcherrimin was not observed only against bacteria Escherichia coli.
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Use of some molecular techniques to metabolic characterization of industrially significant yeasts
Kostovová, Iveta ; Brázda, Václav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Karotenoidy, ergosterol a mastné kyseliny jsou velmi žádané látky využívané v krmivářském, potravinářském a kosmetickém průmyslu. Konvenční zdroje mastných kyselin a karotenoidů jsou závislé na sezónních podmínkách, geografické poloze a na dostupnosti zemědělské půdy, což znesnadňuje pokrýt jejich neustále se zvyšující spotřebu. Velmi slibným řešením je mikrobiální produkce výše uvedených látek pomocí karotenogenních kvasinek, které jsou schopny simultánně produkovat karotenoidy, mastné kyseliny i ergosterol. Předložená disertační práce je zaměřená na molekulární a metabolickou charakterizaci karotenogenních kvasinek a na jejich potenciál pro průmyslové aplikace. Proto první experimentální části práce jsou zaměřeny na kvasinky druhu R. mucilaginosa a R. toruloides, jejich produkční vlastnosti, vliv nutričního stresu a různých zdrojů uhlíku, jakými byly xylóza a glycerol. Kromě podrobné charakterizace jejich produkčních vlastností, byly tyto kmeny také charakterizovány molekulárními metodami, zahrnující sekvenční analýzu ITS1, ITS2 a D1/2 ribozomálního operonu a analýzu mini a mikrosatelitních sekvencí M13 a GTG5. Druh R. toruloides je známý jako vynikající producent mastných kyselin, a proto se v poslední době stal cílovou karotenogenní kvasinkou pro vývoj nástrojů pro jeho genetickou manipulaci. V této práci byly úspěšně připraveny geneticky modifikované klony kmene R. toruloides, nesoucí nadměrně exprimované geny pro diacylglycerol acyltransferázu (DGA1) a glycerol-3-fosfát dehydrogenázu (GPD1). Produkce mastných kyselin u modifikovaných klonů nebyla ve srovnání s původním kmenem vyšší. Proto byla další část práce zaměřená na přípravu nadprodukčních mutantních kmenů připravených náhodnou mutagenezí. Kombinace limitace dusíkem a inhibice produkce karotenoidů vedla k úspěšné selekci robustních mutantních kmenů s nadprodukcí karotenoidů vykazující rezistenci vůči difenylaminu. Poslední část práce se zabývá produkčními vlastnostmi méně známých druhů karotenogenních kvasinek náležící do řádů Sporidiobolales a Cystofilobasidales, ve srovnání s relativně dobře prostudovanými karotenogenními druhy R. toruloides a P.rhodozyma. V této studii byly nejlešími producenty mastných kyselin kmeny S.metaroseus CCY 19-6-20 a C. macerans CCY 10-01-02. Nejlepší producent karotenoidů, kmen R. mucilaginosa CCY 19-04-06, navíc produkoval lykopen, který představoval více než 80 % celkového množství karotenoidů produkovaných tímto kmenem.
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Biotechnological utilization of cellulose wastes by carotenogenic yeasts
Čarnoká, Daniela ; Sikorová, Pavlína (referee) ; Szotkowski, Martin (advisor)
The main objective of the bachelor thesis was to analyze the production of metabolites through the cultivation of selected carotenogenic yeast strains on various waste cellulose substrates. Carotenogenic yeasts are microorganisms capable of utilizing waste cellulose substrates, after suitable chemical pretreatment, as an energy source. Their cultivation leads to the accumulation of biologically active compounds, such as carotenoids, fatty acids, ergosterol, and ubiquinone. The thesis has two main sections: the theoretical part, which encompasses the description of the utilized yeast strains, the produced metabolites, and the waste cellulose substrates, and the experimental part, which details the processing of cellulose waste, yeast cultivation, and the methods employed to determine biomass and metabolite production. The primary waste substrates selected were wood shavings, hemp pellets, and coffee grounds. The study focused on analyzing the production properties of the following yeast strains: Rhodotorula kratochvilae (CCY 20-2-26), Rhodotorula mucilaginosa (CCY 19-4-6) and (CCY 20-9-7), Rhodosporidium toruloides (CCY 062-002-004), Sporidiobolus metaroseus (CCY 19-6-20), Sporidiobolus pararoseus (CCY 19-9-6), and Cystofilobasidium macerans (CCY 10-1-2). The results of the work show that the utilization of cellulose waste as a nutrient source is a possible alternative for the cultivation of carotenogenic yeasts.
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Utilization of dairy industry waste for the production of enriched yeast biomass
Laššová, Erika ; Blažková, Jana (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis deals with the production of microbial biomass of selected carotenoid yeast strains on the waste product of dairy production - whey. The aim of the work was to cultivate selected carotenoid yeast strains on whey medium with the addition of lactase and protease enzymes. The following yeast strains were studied in this work: Rhodosporidium toruloides, Rhodotorula kratochvilovae, Rhodotorula mucilaginosa, Sporidiobolus metaroseus, Cystophilobasidium macerans. The thesis is divided into two main parts. The theoretical part describes the selected yeast strains and their metabolites, the waste substrate used - whey and its use for cultivation of microorganisms. In addition, there is also briefly described the characteristics of enzymes and chromatographic methods of gas and liquid chromatography. The practical part deals with the cultivation of selected microorganisms on the used waste substrate - whey with the addition of lactase and protease enzymes. The cultured biomass was further analyzed by gas and liquid chromatography to determine the content of fatty acids, carotenoids, ergosterol and ubiquinone.
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Biotechnological treatment of poultry industry waste with yeast
Ustiuzhanin, Vladimir ; Sniegoňová, Pavlína (referee) ; Szotkowski, Martin (advisor)
This bachelor's thesis focuses on the cultivation of specific carotenogenic yeast strains using waste substrates from the poultry industry. Yeasts are highly valued for their capacity to degrade complex organic compounds and generate diverse metabolites, including fatty acids, lipids, and carotenogenic pigments. The yeast strains selected for this study were Rhodotorula kratochvilae (CCY 20-2-26), Rhodosporidium toruloides (CCY 62-2-1), Rhodotorula mucilaginosa (CCY 19-4-6), Sporidiobolus salmonicolor (CCY 19-6-4), Sporidiobolus metaroseus (CCY 19-6-20), Sporidiobolus pararoseus (CCY 19-9-6). The work is divided into two parts: theoretical and experimental. The theoretical part provides details on the yeast genera used, a description of the waste substrate, monitored metabolites, and methods applied for their analysis. The experimental part encompasses the cultivation procedures and conditions, biomass processing, metabolite analysis, and the obtained results. Main selected waste substrates were chicken fat and feather hydrolysate. Through a series of laboratory-scale experiments, the most appropriate strains for producing the monitored metabolites and biomass on waste substrates were identified: Sporidiobolus pararoseus a Rhodotorula mucilaginosa. The highest lipid content in the biomass was identified in the strain Sporidiobolus pararoseus.
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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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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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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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