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Controlled production of lipids and lipidic substances by selected yeasts and microalgae
Szotkowski, Martin ; Kráčmar, Stanislav (referee) ; Huelva, Ines Garbayo Nores, University (referee) ; Márová, Ivana (advisor)
Karotenoidy jsou přírodní pigmenty vyskytující se v mikroorganismech jako jsou řasy, kvasinky a sinice. Představují nejrozšířenější skupinu antioxidantů s významným biologickým účinkem. V současnosti vzrůstá zájem o karotenoidy vzhledem k jejich příznivým vlivům na lidské zdraví. Chlorofyly jsou zelená fotosyntetická barviva, která nacházejí uplatnění v potravinářství jako intenzivní zelená barviva. Koenzym Q je znám svým pozitivním vlivem pro správnou funkci řady orgánů v lidském těle. Ergosterol je nedílnou součástí membrán kvasinek a hub. Je to provitamin D2, který je důležitou součástí imunitního systému. Mikrobiální lipidy, nebo také ‚‚Single cell oils‘‘ jsou charakteristické vysokým obsahem zdraví prospěšných nenasycených mastných kyselin, které lze využít ve farmacii či kosmetice. Mikrobiální lipidy jsou dále studovány jako alternativa pro výrobu biopaliv. Dizertační práce byla zaměřena na studium a možnosti optimalizace produkce lipidů a lipidických látek vybranými kmeny karotenogenních kvasinek, mikrořas a sinic. V rámci práce byly testované kvasinky rodu Rhodotorula, Rhodosporidium, Cystofilobasidium a Sporidiobolus podrobené kultivacím na sérií médií s různými C/N poměry v rozsahu 13 až 100, obsahujících upravené odpadní substráty z potravinářského průmyslu. Vybrané kmeny byly poté kultivovány v bioreaktorech v médiu obsahujícím kombinaci odpadních substrátů. Kultivace mikrořas rodu Desmodesmus, Scenedesmus, Chlorella, Coccomyxa, Chlamydomonas, Botryococcus se zabývaly optimalizací jednotlivých komponent média a aplikací různých stresů s cílem navýšení produkce studovaných metabolitů. V rámci experimentů s extrémofilní mikrořasou Coccomyxa byly provedeny pilotní velkoobjemové kultivace v otevřených nádržích. V závěrečné části byl provedeny pilotní screeningové a velkoobjemové bioreaktorové experimenty zaměřené na možnosti kokultivace karotenogenních kvasinek a mikrořas. Testované kmeny kvasinek byly s rozdílnou úspěšností schopny utilizovat média obsahující hydrolyzované odpadní substráty. Nejlepším kmenem byl Sporidiobolus pararoseus, který na médiích dosahoval nejvyšších produkcí biomasy i sledovaných metabolitů. Z testovaných odpadních substrátů byla nejlepší kombinace odpadního fritovacího oleje a hydrolyzátu kávové sedliny. Úspěšná optimalizace složení hlavních komponent minerálního média vedla k zvýšené produkci studovaných metabolitů. Největší vliv měl optimální poměr P/N a aplikace oxidačního stresu. Nejlepších výsledků dosáhly mikrořasy rodu Desmodesmus a Scenedesmus. Velkoobjemové kultivace Coccomyxy onubensis potvrdily rezistenci kultury proti kontaminaci vnějšími vlivy a schopnost růstu za vysoké teploty a intenzity světelného záření. Kokultivační experimenty potvrdily schopnost symbiotického růstu kvasinek a mikrořas. Nejlepších výsledků dosahovaly všechny testované kvasinky s mikrořasami rodu Demsodesmus a Scenedesmus a v menší míře i rodu Coccomyxa.
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Optimization of cultivation of carotenogenic yeasts on mixed waste substrates
Holub, Jiří ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The master thesis addresses the issue of cultivation of selected strains of carotenogenic yeasts on waste materials of the food industry using a laboratory bioreactor. Carotenogenic yeasts are able to produce highly valuable metabolites during cultivation, which are located predominantly in the lipid part of the cells. Particularly, they are carotenoids, ergosterol, coenzyme Q and fatty acids. The thesis is divided into two main parts, the theoretical part and the practical part. The theoretical part describes individual yeast strains, types of waste materials, produced metabolites and methods of their analysis. The experimental part deals with the processing of waste materials of the food industry, specifically animal fat, whey and spent coffee grounds into the form of substrates usable as nutrition sources for yeast cultivation. Furthermore, cultivations focused on the recovery of the monitored metabolites and their analysis by using HPLC/PDA and GC/FID assemblies were studied as well. The yeast strains Rhodotorula mucilaginosa (CCY 19-4-6), Rhodotorula kratochvilae (CCY 20-2-26), Rhodosporidium toruloides (CCY 062-002-001), Sporidiobolus pararoseus (CCY 19-9-6) a Cystofilobasidium macerans (CCY 10-1-2) were used in this work. As one of the best producing strains Sporidiobolus pararoseus (CCY 19-9-6) was found, which achieved very high productions of carotenoids, coenzyme Q and ergosterol.
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Metabolic adaptation of carotenogenic yeasts to nutritional stress induced by selected wastes of the food industry
Plhalová, Žaneta ; Šimanský, Samuel (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis deals with the production of carotenoids and lipids by selected strains of carotenogenic yeasts cultivated on waste substrates of the food industry. The production properties of the yeast strains Sporidiobolus pararoseus, Sporidiobolus metaroseus, Cystofilobasidium macerans, Rhodotorula mucilaginosa, Rhodotorula kratochvilovae and Rhodosporidium toruloides were studied. The cultures were cultivated by using waste products of food industry: waste frying oil, coffee oil which was obtained by extraction from coffee grounds and waste animal fat from rendering plants. The work consists of two main parts. The theoretical part describes the individual genera of yeasts, monitored metabolites, waste products and methods of analysis of monitored metabolites. The experimental part is focused on the processing of used waste substrates, cultivation of yeasts, acquisition and processing of biomass and analysis of metabolites. Rhodosporidium toruloides and Sporidiobolus pararoseus strains were the most suitable for the production of carotenoids on waste substrates. The highest production of lipid substances was measured in the strain Sporidiobolus pararoseus.
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Simultaneous co-cultivation of selected strains of carotenogenic yeasts and autotrophic bacteria
Blažková, Jana ; Němcová, Andrea (referee) ; Szotkowski, Martin (advisor)
The submitted diploma thesis was focused on the study of co-cultivation of selected microorganisms, which were carotenogenic yeasts and cyanobacteria. The production of selected metabolites was compared in these co-cultivations. The main metabolites monitored were carotenoids, sterols, coenzyme Q10, chlorophylls and lipids. Furthermore, this work focused on the study and possibilities of optimizing the production of lipids and lipid substances in selected strains of carotenogenic yeasts and cyanobacterial species. The theoretical part is focused on the description of yeasts, especially carotenogenic yeasts, cyanobacteria and the chemical composition of the produced metabolites. Microorganisms such as yeast and cyanobacteria contain carotenoids, which are natural pigments and are classified as antioxidants. As antioxidants, they have significant biological effects, such as effects on human health. Coenzyme Q has a positive effect on the functioning of organs in the human body. Chlorophyll is widely used in the food industry as a green dye. Lipids produced by microorganisms contain a large amount of unsaturated fatty acids, which is currently used in cosmetics or pharmacy. The theoretical part also described the individual analytical methods by which the production of the monitored metabolites could be determined. The experimental part is focused on the production of carotenoids, sterols, coenzyme Q10 and chlorophyll, which were determined by HPLC, lipids and fatty acid profile were determined by GC. The determined metabolites are monitored in different types of co-culture partners (carotenogenic yeast and cyanobacteria) in media with different additions of macroelements (P, N and Mg). This was followed by a co-cultivation experiment using waste oils (frying and coffee oil) and a study of the effect of waste oils added to co-cultivations. Co-cultivation experiments confirmed the ability of carotenogenic yeasts and cyanobacteria to grow together. The best results were obtained with Rhodosporidium toruloides and Anabena torulosa, Rhodosporidium toruloides and Arthrospira maxima.
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Optimization of extraction of metabolites produced by selected strains of microalgae and carotenogenic yeast.
Obračaj, Jan ; Holub, Jiří (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis is focused on the production of microbial biomass using selected strains of cyanobacteria and carotenogenic yeasts. Subsequent preparation of extracts and study of their stability under different temperature conditions with the addition of the antioxidant tocopherol. In the experiment, 2 series of extracts with hexane and ethanol were prepared. The theoretical part of the work describes the studied metabolites carotenoids, chlorophylls, ubiquinone, ergosterol and microbial lipids. Furthermore, the used strains of cyanobacteria and carotenogenic yeasts are described here. Finally, methods for extraction and analysis of metabolites are described. The experimental part describes the used cultivation techniques, preparation of microbial extracts and used analytical methods. The results part of the work then contains a summary of the results of long-term stability tests of extracts. The results of the work clearly confirm the temperature dependence of the stability of individual metabolites. This phenomenon was most evident in chlorophyll and carotenoid pigments. Hexane appears to be the best solvent in terms of the stability of the substances used. The results of this work provide important information in terms of the economics of storage of these valuable substances usable in the food, pharmaceutical or feed industries.
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Determination of ergosterol content in brewing materials
Matyáš, Martin ; Ing.Pavla Kožušníková, Ph.D. (referee) ; Benešová, Karolína (advisor)
Presented bachelor’s thesis is focused on determination of ergosterol content in barley and malt. In theoretical part the presence of ergosterol in barley and malt has been elaborated, the purposes of its determination, detailed overview of previously applied methods of extraction from samples of barley and malt as well as other biological materials and methods of quantification. Practical part is focused on optimizing of extraction and determination of ergosterol by high performance liquid chromatography with diode array detection (HPLC – DAD).
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Biotechnological valorization of some food wastes by yeasts
Majdloch, Robert ; Bendová, Agáta (referee) ; Márová, Ivana (advisor)
This bachelor thesis deals with biotechnological valorisation of waste substrates, namely waste fat, whey and feathers using carotenogenic yeast. Waste substrates serve as alternative source of nutrients for subsequent cultivation, thanks to this, their utilization and subsequent production of carotenoids, ergosterol, coenzyme Q and lipids occurs. The theoretical part contains the information of selected strains of carotenogenic yeast, such as physiological and growth properties. Their metabolic processes are described together with produced substances. The analytical methods for the determination of substances are described as well. The experimental part focuses on the solution of flask and bioreactor cultivations of four selected strains (Rhodotorula kratochvilovae, Rhodotorula mucilaginosa, Cystofilobasidium macerans a Sporidiobolus pararoseus) on glucose, glycerol and waste substrate at different C/N ratios (13, 25, 50 and 100). Furthermore, individual procedures of waste substrate processing, procedures solving isolation and determination of selected carotenoid substances, which were analysed by HPLC/DAD. Lipid content and their percentage amount were determined by GC/FID. The results of flask and bioreactor cultivations show that the strains have different production at individual C/N ratios. Rhodotorula mucilaginosa strain was selected as the best producer of carotenoids and ergosterol. The highest increases in biomass were achieved by Rhodotorula kratochvilovae strain. Lipid production was the best in the Sporidiobolus pararoseus strain.
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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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