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Characterization of waste rice substrates and their potential utilization by microorganisms
Kapar, Jiří ; Duroňová, Kateřina (referee) ; Márová, Ivana (advisor)
The aim of this bachelor thesis was to characterize some cereal substrates. Based on the results of group parameters the most suitable substrate for cultivation of model industrial microorganisms was proposed. Analysis of total phenolics, reducing sugars and total sugars was performed spectrophotometrically. Using high-performance liquid chromatography with refractometry index detection content of individual mono-and disaccharides was measured. Saccharides were extracted from cereal substrates by water and then acid hydrolysis was applied. Two rice substrates were then used as nutrition sources for cultivation of two model industrial microorganisms: yeast Rhodotorula glutinis and bacterium Bacillus subtilis. Before use to production media substrates were processed by chemical and enzyme hydrolysis and analyzed. Production of microbial biomass in media with modified waste substrates was evaluated.
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Production of lipid substances by Metschnikowia yeasts grown on some waste substrates
Gonová, Dominika ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
Oleaginous yeasts posses the ability to accumulate increased amount of lipids under appropriate conditions. These microbial lipids vary in the composition of fatty acids which results in their wide application in the biotechnological industry. This master thesis focuses on the lipid production and fatty acids composition from waste substrates by the yeasts Metschnikowia depending on various cultivation conditions. The influence of temperature, the ratio of carbon and nitrogen in medium, and the concentration of different carbon sources was studied. The cheap and easy available waste substrates as glycerol and animal fat were used for the cultivation. The production characteristics of the yeasts were monitored by various technique including gas chromatography, Raman spectroscopy and fluorescence microscopy FLIM. Moreover, the partial optimalization of the pulse field gel electrophoresis was applied in order to characterize the karyotype of the yeasts Metschnikowia. All the studied strains were able to use the waste substrates and at the same time to produce lipids. The amount of lipids and mainly their compositions vary depending on the yeast strain and on the culture conditions. Nevertheless, the ability of the yeasts to produce significant amount of unsaturated fatty acids by manipulation of culture conditions was proved. The maximum lipid yield was achieved by M. pulcherrima 149 on glycerol medium and by M. andauensis 129 on medium containing waste animal fat.
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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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Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
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Application of microbial oils in cosmetics
Reichertová, Klára ; Uhlířová, Renata (referee) ; Němcová, Andrea (advisor)
This bachelor thesis is focused on the application of microbial oil in a cosmetic product. Eight strains of yeasts of the genus Metschnikowia were examined, namely Metschnikowia pulcherrima 145, Metschnikowia pulcherrima 147, Metschnikowia pulcherrima 149, Metschnikowia andauensis 129, Metschnikowia andauensis 1241, Metschnikowia sinensis 1244, Metschnikowia zizyphicola 1247 and Metschnikowia shanxiensis 1250 for production of microbial oil. Substrates made of technical hemp (hemp flour, hydrolyzate and a mixture of leaves and flowers) were used for cultivation. The cultivation was performed at reduced temperature with the advantage of lipid production. The composition and ratio of fatty acids were determined by gas chromatography According to these resulst was choosen the best lipid producer and the most abundant substrate. This was the strain Metschnikowia zizyphicola 1247 and the substrate of hemp flour. At the end of the practical part was prepared a cream, into which the microbial oil was mixed. Finally, an evaluation of the oil with regard to its use in cosmetics and a comparison with vegetable oil with the same fatty acid profile are given.
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Employment of bacterium Tepidimonas taiwanensis for production of polyhydroxyalkanoates on waste substrates
Vidláková, Michaela ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on screening PHA production using thermophilic bacterium Tepidimonas taiwanensis and on the study of possible use of grape pomace, molasses and waste paper as a cheap carbon substrate for culturing the characterized bacteria. At first, testing of basic cultivation parameters was performed, including carbon substrate concentration, oxygen availability, ability to utilize nitrogen sources and selected disaccharides. PHA production from waste substrates was tested in three ways. In the first, pre-prepared solids-free hydrolysates from raw materials were used as the carbon source. The second and third procedures were performed by dosing waste materials directly into the mineral media, which differed only in the presence or absence of the enzyme preparation enabling release of fermentable sugars. The most intensive increase in culture and the highest production of PHA was recorded on grape hydrolyzate. The biomass concentration in this sample reached up to 4.8 g/L with a content of 59 % PHA. On the other hand, the addition of grape marc directly to the production medium did not work at all, which was probably due to the presence of a large amount of inhibitory substances from the pomace. The situation was similar with molasses and waste paper, where the bacterium Tepidimonas taiwanensis was able to grow and possibly produce PHA only to a small extent. The work also managed to characterize the effect of temperature and pH on the activity of the enzyme cocktail Viscozyme L and to determine the temperature and pH optimum PHA synthase of the bacterial strain Tepidimonas taiwanensis in the cell lysate.
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Study of production properties of Metschnikowia yeasts
Chadimová, Markéta ; Szotkowski, Martin (referee) ; Němcová, Andrea (advisor)
Metschnikowia yeasts are able to produce under different conditions different amounts of lipids, which have remarkable uses in biotechnology and industry. This bachelor thesis is focused on the study of cultivation conditions under which yeasts produce the most lipids and also deals with which fatty acids these triacylglycerols are composed of. The influence of cultivation conditions on the amount of biomass is also monitored. Five yeast strains were examined, namely M. pulcherrima 145, M. pulcherrima 147, M. pulcherrima 149, M. andauensis 129 and M. chrysoperlae 1158. Cultivation media with different C/N ratios, containing glucose or cheap waste substrates (glycerol, coffee and waste fat) were used for cultivation. The cultivations were performed at several temperatures and then the amount of grown biomass was determined, and the composition and ratio of fatty acids were determined by gas chromatography. As yeast that produced the most biomass per time was determined the yeast M. chrysoperlae 1158, which after 336 hours of growth at 15 °C was able to produce 10,66 g/l of biomass on a medium with a ratio of C/N = 100. The highest amount of lipids was obtained with yeast M. andauensis 129 at 8 °C on medium with a ratio of C/N = 100 (21,57 % lipids in dry matter). If biomass production is also taken into account, the best results are obtained by the yeast M. chrysoperlae 1158 at a temperature of 11 ° C on a medium with a ratio of C/N = 100 with a biomass production of 10,15 g/l and 19,58 % lipids in dry matter. This yeast was therefore further cultivated on waste substrates.
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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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Random mutagenesis and selection of red yeast mutants capable to utilize particular waste substrates
Čačková, Katarína ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.
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Use of yeasts of the genus Metschnikowia for the lipid production
Švitková, Bibiána ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
The oleaginous yeasts have an ability to accumulate an increased number of lipids, under certain circumstances. These microbial lipids differentiate in the number of fat acids present, which enables their wide application in biotechnological industry. This master’s thesis is aimed on lipid production, number of the fat acid groups present, and squalene production by Metschnikowia yeasts, based on the cultivating conditions. Biomass and lipid production was observed in separate cultivation media, with the addition of the different waste substrates. Production properties were observed by method of the gas chromatography. For the squalene production observation, a HLPC method was chosen. All production groups were able to accumulate lipids on the waste substrate, although in different values. These values were very individual, especially in the areas of the specific groups and growth on the given substrate. The lipid composition was different, which was caused by differences in the waste substrates. With regards to the squalene production – the yeasts from the Metschnikowia family were not able to produce squalene in the presence of the terbinafine and its increasing concentration. Therefore, the same procedure was chosen, as it was for the Yarrowia lipolytica yeast, with the difference in the sterol synthesis, however squalene was still not produced this way.
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