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Use of carotenogenic yeasts to production of lipid soluble metabolites
Mariničová, Veronika ; Hlaváček, Viliam (referee) ; Márová, Ivana (advisor)
Carotenoids are fat-soluble chemical compounds that occur as natural pigments in many plants and protect them from sunlight. Lipids are also essential lipophilic substances and they are part of biomembranes. Their main function is primarily to serve as a power supply for the cell, protective function and thermal protection against adverse environmental influences. This bachelor thesis deals with cultivation of selected carotenoid yeast genes, subsequent isolation of carotenoids and other lipid substances, which can be used as a source of potentially beneficial substances for the pharmaceutical or cosmetic industry. The theoretical part deals with the description of carotenoid yeasts, chemical composition and biosynthesis of the metabolites produced, and description of the methods used for their determination. The experimental part is focused mainly on the production of lipid substances by various strains of yeasts using cheap waste substrates and the application of exogenous stress (nutritional stress) to the biotechnological overproduction of selected metabolites using the modification of the production medium. The content of carotenoids, ergosterol and coenzyme Q was analyzed by high performance liquid chromatography with a PDA detector. The lipids accumulated in yeast cells were determined by gas chromatograph with a FID detector. In this work the strains of Sporobolomyces pararoseus, Sporobolomyces metaroseus, Rhodotorula glutinis and Cystofilobasidium infirmominiatum were studied. As waste substrates and carbon sources were used glycerol, which is produced as a waste product in the production of biofuels and whey as an unusable product in dairy technology. The best production on waste substrates was observed in the strains Rhodotorula glutinis and Cystofilobasidium infirmominiatum.
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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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Study of carotenogenic yeasts doring growth by using advanced instrumental techniques
Vaněk, Martin ; Breierová, Emília (referee) ; Márová, Ivana (advisor)
This work is dealing with application of advanced fluorescence techniques for gaining knowledge about culture development during fermentation of red yeasts. Flow cytometry was used for auto-fluorescence measurement a carotenoids quantitation. It was resolved that while carotenoids are stored mainly in membranes the technique was feasible. If red yeast starts to accumulate carotenoids into lipid bodies mainly throughout the course of stationary phase, then the method starts to fail. Flow cytometric method using cell size measurement and light scatter for lipid quantitation was proved as applicable, too. However, it works only if cells are not starved. Individual calibration for each species is needed for elimination inter-species variations of intracellular structures. Fluorescence lifetime imaging microscopy was also used for studying of red yeast. Inherent ability to resolve different fluorescent species of the same molecule, which arise due to different molecular environment, helps with quantitation of cellular lipidic structures changes through the course of fermentation. Increase in the levels of carotenoids and/or rigidity of membranes was found as mechanism of protection during metabolic shifts, when intracellular content is vulnerable to damage.
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Condition dependence of secondary ornamentation and mate choice in Grey Partridges
Gabrielová, Barbora ; Albrecht, Tomáš (advisor) ; Sedláček, Ondřej (referee)
This study focuses on relationships between individual condition, measures of ornament expression and mate choice in the Grey Partridge (Perdix perdix). It is based on the correlative data as well as results of manipulative experiments. I found positive correlation between the size of melanin- and carotenoid-based ornaments in partridges. The size and the redness of the carotenoid skin patch, and the size of the melanin-based feather ornamentation were associated with several measures of immunity and overall body condition (complement activity, ratio of immature erythrocytes, heterophil/leucocyte ratio, response to novel antigen). Furthermore, data from natural populations indicate that the expression of carotenoid-based ornamentation is associated with sexual attractiveness (measured as the ability to obtain a social mate) in partridges. Data from captive individuals indicate that carotenoid supplementation improves the redness of carotenoid-based ornament, while the stimulation of the immune system under increasing concentrations of supplemented carotenoids has no measurable effect on condition and physiological state. This seems to contradict predictions of the 'Carotenoid maintenance hypothesis'. Overall, results better understanding the role of carotenoid and melanin-based ornamentation in signaling...
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Response of different genotypes of Zea mays L. and Vicia faba L. treated with brassinosteroids to water deficit
Kuklíková, Radka ; Rothová, Olga (advisor) ; Hnilička, František (referee)
This work monitors the response of plants to the application of a solution of synthetic brassinosteroid (5-fluor-3α,17β-dihydroxy-5α-pregnan-6-on) under drought stress conditions and subsequent restoration of water availability. The response of two plant species was monitored under six-day period of cessation of watering and under six-day period of renewed optimal water supply. The experiments were carried out on plants with different type of photosynthesis - C3 plant Vicia faba L. and C4 plant Zea mays L.. For both plant species, three varieties/genotypes were used. Photosynthetic, morphological and developmental characteristics were monitored. The brassinosteroid treatment influenced more the morphological than the photosynthetic characteristics. The content of photosynthetic pigments was influenced more than the chlorophyll fluorescence parameters. The differences in morphological characteristics between individual varieties/genotypes were observed. The response to the application of brassinosteroid was observed especially for drought-sensitive variety/genotype. Application of brassinosteroid caused increase of the dry mass of leaves and roots, the height to leaf , the height of the whole plant and its increment under stress and post-stress periods in both plant species. The content of...
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Inter- and intraspecific variability in Vicia faba L. and Zea mays L. response to water deficit
Fridrichová, Lenka ; Holá, Dana (advisor) ; Zelenková, Sylva (referee)
This work monitors the response of plants to water deficit and subsequent restoration of water availability with the aim to expand our knowledge about plant behaviour during and after drought stress from the perspective of two plant species with a different type of photosynthesis. A particular attention was paid to the intraspecific differences and their possible changes during stress and post-stress periods. The plant response was monitored as the changes of selected photosynthetic, morphological and developmental characteristics caused by the cessation of watering for six days followed by a six-day period of renewed optimal water supply. The experiments were carried out on plants with different types of photosynthesis - C3 plant Vicia faba L and C4 plant Zea mays L.; for both plant species, three varieties / genotypes were monitored. The response of both plant species to insufficient water supply differed by the extent of changes in relative water content (RWC), chlorophyll fluorescence parameters, the dry mass of individual leaves (developed during the stress period), the ratio between the aboveground dry mass and dry mass of roots. In drought-stressed plants, downward trend in RWC was observed, as well as the increase of the minimum yield of chlorophyll fluorescence in dark-adapted leaves (F ),...
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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 selected fluorescence techniques to study of yeasts and yeast metabolites
Mikheichyk, Nadzeya ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The scope of thesis was the optimization of methods for the study of yeast and their metabolites using flow cytometry and fluorescence microscopy. Red yeasts are characterized by overproduction of carotenoids and lipids, which are used in food, pharmaceutical and feed industries. Currently, intensive research is being carried on to find appropriate microbiological alternatives for synthesis of these substances. Present thesis is focused on selected yeast genera: Rhodotorula, Sporobolomyces, Cystofilobasidium and strain Phaffia rhodozyma. Yeasts were cultivated on different nutrient media, in which glucose was used as a nutritional source, and also on glycerol and whey as waste material. In two strains - Cystofilobasidium macerans and Rhodotorula mucilaginosa growth characteristics were determined on a synthetic glucose production medium. All studied strains were able to use waste substrates as a source of nutrients. Some of the strains displayed increased production of carotenoids, and, additionally, in some cases also relatively high production of lipids. In classical cultivation in lipid and glucose medium supplemented with vitamins the best production characteristics displayed Rhodotorula glutinisstrain. In glycerol medium the highest amount of carotenoids and lipidic substances produced Sporobolomyces shibatanus strain. Strain Sporobolomyces roseus showed the best production characteristics on whey as the main source of carbon. The results show use of whey and glycerol seems like appropriate option for potential carbon source to cultivate carotenogenic yeasts and production of carotenoids and selected lipidic substances as products with higher added value. Further optimization of nutrient medium on the given substrates is needed for higher production of selected metabolites. Fluorescence microscopy and flow cytometry have proved to be suitable options for determination of the observed metabolites in the cells, their amount and viability.
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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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OPtimization od cultivation od microalgae cultures in photobioreactors
Byrtusová, Dana ; Márová, Ivana (referee) ; Kočí, Radka (advisor)
Presented diploma thesis is focused on the optimisation of Haematococcus pluvialis cultivations in different photobioreactors and on biotechnological production of astaxanthin. Theoretical part summarized the knowledge about optimal growth and production conditions of secondary metabolites. Followed research was focused on actual cultivation systems and on the possibilities of metabolite and nutrient monitoring. In experimental part the growth characteristic of the strain from Březova nad Svitavou (HMP-CCALA 375) was analyzed under optimal cultivation conditions on white and red light. During culture growth the profile and the concentration of carotenoid pigments were determined. The best yield of biomass was achieved in the cultivation on white light (0,939 g/l),carotenoids lutein and -carotene were observed as dominant pigments. In the next experiments optimal growth medium, temperature and light intensity were determined for cultivations of four chosen HMP strains from Germany, America, Africa and Switzerland. The most suitable cultivation medium was found BBM, oppositely the worst results were obtained with BG11. In previous experiments cultivation temperature 22 °C was determined as optimal value for comparative strain HMP – CCALA 375. Selected four strains were cultivated at 22 °C, as well as at 25 °C. Higher temperature was more optimal mainly for Switzerland, German and Africa strains. By analyzing of light intensity influence, it was found that the best increase of biomass was induced by the adaptation of culture on lower illumination (50 µmol photones•m^-2•s^-1) followed by higher light intensity (100 - 150 µmol photones•m^-2•s^-1). HMP from Switzerland showed the best growth results during all cultivation experiments, so this strain could be perhaps useful for industrial production of astaxanthin. In the last part of work, the influence of stress conditions on astaxanthin production by strain from Březova nad Svitavou (HMP – CCALA 375) was studied. Followed stress factors were used: high intensity of light (1 000 µmol photones•m^-2•s^-1), low nitrogen concentration (32,96 mg/l), addition of sodium chloride (0,5%), influence of sodium acetate (2,2 mM) and combination of sodium chloride and sodium acetate (0,5% NaCl, 2mM NaAc). Due to strong illumination (1 000 µmol photones•m^-2•s^-1) the best yield of astaxanthin was obtained (more than 20 mg/g). According to literature [103, 105] significant amount was also observed by addition of sodium acetate (9,2 mg/g). Oppositely minimal astaxanthin production was showed in presence of salt stress (3,8 mg/g). In followed experiments should be studied the influence of stress combinations on HMP – CCALA 375 strain as well as on other suitable strains of H. pluvialis with the aim to achieve the maximal yield of astaxanthin significant for large scale cultivation.
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