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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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Use of selected fluorescence techniques to characterization of microbial cells
Vaněk, Martin ; Pekař, Miloslav (referee) ; Márová, Ivana (advisor)
Carotenoids are one of the most abundant natural pigment. Furthermore, they have great potential for wide industrial utilization. Nowadays, prefered (and cheaper) synthetic method counters progressively improved biotechnological production. Far more cheaper production should be reached via implementing methods of on-line optimization, such as flow cytometry. In this thesis the dependence of yeasts autotofluorescence intensity on carotenoids concentration in dry mass was studied. The dependence was established by means of linear regression. Moreover, the dependence between light scatter and the amount of dry mass was established by means of model utilizing forward scatter, side scatter and number of cells in unit volume. Yeasts' cells was also studied under the lenses of laser scanning confocal microscope. The goal was to localize carotenoids inside cells. Experiments undertaken suggest that they are localized inside the lipidic granules, but more experiments are required for a proof.
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Production of carotenoids grown on waste fat
Holub, Jiří ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
Carotenoids are lipid-soluble pigments which are contained in plants and microorganisms. Carotenoids are known for their antioxidant effects. Lipids are predominantly non-polar substances, which are essential and present in all types of organisms. The research was made by theoretical and practical form. It was dealing with production of carotenoids pigments and different lipophilic substances by yeasts of Rhodotorula glutinis, Rhodosporidium toruloides and Sporidiobolus metaroseus, cultivated in media containing waste fat or glycerol. Further, methods for obtaining lipid substances from microorganisms and with further analysis of samples by gas or liquid chromatography were optimized. The main task of the research was to find out which types of media and yeasts are the most suitable for production of lipid substances. For production of lipid substances in glycerol media, as the best producing strain Rhodosporidium toruloides was found, which simultaneously reported the highest concentrations of carotenoids on fatty and fatty hydrolyzed media.
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Miniaturized techniques for analysis of industrial yeasts
Obalil, Jiří ; Kočí, Radka (referee) ; Klepárník,, Karel (advisor)
Carotenoids are natural pigments that have antioxidation and antimutagenic abilities. They are produced with the help of new technological methods. For example, carotenoid yeast Rhodotorula glutinis produces -carotene with the yield of up to 6 – 10 mg/g of the dry substance. The method of the mass spectrometry with the nanoelectrospray in the positive mode was optimized for the determination of -carotene, lycopene and astaxanthin in this project. Ionizing voltage of 4 kV and the sample flow rate of 15 – 80 nl/min through the spray silica fused capillary with the internal diameter of 25 µm were found to be the optimum parameters of the analysis. A mixture of chloroform with the addition of ammonia was used as a spray solvent for both standard and cellular samples. During the process of ionization by nanoelectrospray, -carotene and lycopene form cation radical [M] • + with the molecular mass to charge ratio (m/z) of 536, while asthaxanthin forms the protonated molecule [M + H]+ with the m/z of 597. The partial lysis of individual Rhodotorula glutinis cells was demonstrated under microscope in the organic solvents tetrahydrofuran and dimethylsulfoxide. Chloroform, acetone, acetonitrille, methanol and isopropanol did not affect the cells after a 15 min treatment.
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Production of carotenoids by red yeasts grown on some waste substrates
Kubáčková, Martina ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the most widespread and abundant classes of natural pigments with utilised in food industry, pharmacy and cosmetics. Presented work was realized as a comparative study of some substrates for cultivation of red yeast Rhodotorula glutinis, which produces carotenoid pigments. Yeasts were cultivated in different media with several waste substrates (for example potato fiber, apple waste, cereals, grains, etc.) as nutrition sources. To some substrates hydrolytic enzymes isolated from the fungi genus Fusarium were added. Majority of waste substrates were acceptable for cultivation. However, the best conditions for production of carotenoid enriched biomass (6,4 g/l of biomass enriched 3,2 mg /g of beta-carotene) exhibited media with hydrolytic fungal enzymes.
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Use of microscopy and flow cytometry to molecular characterization of carotenogenic yeasts.
Vacková, Hana ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The aim of presented bachelor thesis is to optimize the methods for study of yeasts using flow cytometry and fluorescence microscopy. The theoretical part deals with the structure of yeast cells, principles and application of flow cytometry and fluorescent microscopy. Use of microscopy and flow cytometry in study of pigmented microorganismswas reviewed too. In the experimental part methods optimization was performed on yeasts Cystofilibasidium capitatum, Rhodotorula glutinis, Sporobolomyce sroseus, Sporobolomyces shibatanus. Further, the growth curve and the viability of yeasts under the influence of stress factors (freezing and the heat shock 60 C) was measured. Cystofilobasidium capitatum has shown to be more resistant to stress than Rhodotorula glutinis. It was found that there is the possibility of using methylene blue instead of propidium iodide in yeast cell analysis by flow cytometry.
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Regulation of production of lipids and lipid compounds in yeasts
Rapta, Marek ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
Oleogenic yeasts under appropriate conditions produce and accumulate lipids and lipid-soluble metabolites in high amounts. This attribute is characteristic also for red yeasts that except lipids accumulate also carotenoids – natural pigments used in food industry and dietary supplements. The aim of this diploma thesis was designed as a comparative screening study of production properties of strains Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces roseus and Sporobolomyces shibatanus. Choosen carbon sources were glucose and glycerol as waste by-product in biofuel industry. The best production properties were found in Cystofilobasidium capitatum and Rhodotorula glutinis. These two strains produced increased amounts of lipids as well as higher amounts of carotenoids. Strains were tested by FTIR spectroscopy that enables high-throughput, uncomplicated and accurate analysis.
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Microbial production of carotenoid pigments using waste substrates
Němcová, Andrea ; Ing.Martina Čarnecká, Ph.D. (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments produced by bacteria, yeasts, filamentous fungi and plants. They exhibit significant biological effects and are widely used in the food industry, pharmacy and cosmetics. The aim of this diploma thesis proposed as a comparative study was regulation of carotenoid and ergosterol production in red yeasts using several waste substrates as whey, corn germs, wheat, apple fiber and pasta. To selected production media extracellular hydrolytic enzymes degrading polysaccharide were added. These enzymes were obtained from the cultivation media of four fungal strains. In this study three carotenogenic yeast strains were used: Rhodotorula glutinis, Sporobolomyces roseus and Cystofilobasidium capitatum. All strains were cultivated simultaneously and changes in biomass and carotenoid production in different production media were monitored and compared. As the best waste substrate apple fiber was utilized, particularly in Rhodotorula glutinis, which exhibited mainly biomass production increase. In Sporobolomyces roseus increased production of biomass and carotenoids have been reported in media with hydrolyzed fiber and pasta as well. Beta-carotene production in this strain reached 4776,38 mg/g of dry weight. The strain Cystofilobasidium capitatum exhibited in waste media a decerase of biomass production accompanied with increased production of carotenoids, especially in wheat mush and pasta medium hydrolyzed by enzyme preparative from Aureobasidium pullulan. It can be concluded that hydrolyzed waste substrates are very hopeful as cheap nutrient sources for yeast strains producing carotenoids and ergosterol. Nevertheless, further study of substrate processing for individual strains is needed.
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Characterization of individual phases of cultivation of red yeasts
Valentová, Lucie ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This bachelor thesis deals with the problematic of metabolic activity and morphology of several biotechnologically significant red yeasts - Rhodotorula glutinis, Sporobolomyces shibatanus and Phaffia rhodozyma during the cultivation time. These yeasts were cultivated on glucose medium, during the cultivation samples were taken for the analysis of produced metabolites. The amount of -carotene, astaxanthin, coenzyme Q and ergosterol were monitored. The amount of biosynthesised lipids and fatty acid profile was also investigated. HPLC/PDA and GC/FID were used for the analysis of these metabolites. Yeasts were cultivated 56 hours (they reached early stationary phase). The highest production of carotenoid pigments showed Phaffia rhodozyma (1856.58 g/g), 42 % of this amount represents -carotene. The highest lipids biosynthesis efficiency was reached by Rhodotorula glutinis. To study the inner structure of yeast cells the Fluorescence Life-time Imaging was used, the Autofluorescence of carotenoids and fluorescence of lipid bodies induced by lipophilic dye Nile red was observed. This method is very useful for imaging cell inner structure (membranes, some kinds of organelles). The changes that the cell undergoes during its evolution could be quantified too. The relationship between the level of carotenoid concentration, membrane rigidity and metabolic shift was found.
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Production of carotenoids grown on waste fat
Holub, Jiří ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
Carotenoids are lipid-soluble pigments which are contained in plants and microorganisms. Carotenoids are known for their antioxidant effects. Lipids are predominantly non-polar substances, which are essential and present in all types of organisms. The research was made by theoretical and practical form. It was dealing with production of carotenoids pigments and different lipophilic substances by yeasts of Rhodotorula glutinis, Rhodosporidium toruloides and Sporidiobolus metaroseus, cultivated in media containing waste fat or glycerol. Further, methods for obtaining lipid substances from microorganisms and with further analysis of samples by gas or liquid chromatography were optimized. The main task of the research was to find out which types of media and yeasts are the most suitable for production of lipid substances. For production of lipid substances in glycerol media, as the best producing strain Rhodosporidium toruloides was found, which simultaneously reported the highest concentrations of carotenoids on fatty and fatty hydrolyzed media.
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