|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
Cultivation of carotenogenic yeasts on waste substrates usinf solid state fermentation
Rapta, Marek ; Petrik, Siniša (referee) ; Márová, Ivana (advisor)
Semi-solid fermentation is an eco - friendly technique more and more used for production of significant metabolites or enriched biomass at low entrance cost and low comsumption of water. The problem of this technique might be the right choice of microorganism able to grow and produce at low water activity. This work is a pilot study of three red yeast strains – Rhodotorula glutinis, Cystofilobasidium capitatum and Sporobomomyces roseus cultivated by semi-solid state fermentation (semi-SSF). Yeasts were cultivated in series of production media with gradual reduction of cultivation water content. Pasta and hydrolyzed pasta were used as source of sacharides, glucose served as the carbon source in control media. All studied strains we able to grow and produce observed lipidic metabolites also at low water contents. Production of carotenoids and sterols was higher in semi-solid media. Cystofilobasidium capitatum strain was identified as the best producer of carotenoids and biomass. This strain was cultivated on hydrolyzed pasta media with 40% water content. Semi-SSF seems to be an adequate technique for selection of strains having over-productive properties. Observed metabolites production in yeast cells can be optimized by choosing the appropriate production media and water activity.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
Production of carotenoids and enriched yeast biomass on rape-seed waste substrates
Mikheichyk, Nadzeya ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
Carotenoids belong to the group of the most widely used natural pigments with significant biological effects. Recently, microbiological production of carotenoids prior synthetic production is examined. The aim to the study was to analyse the properties of carotenogenic yeasts on waste rapeseed substrate. The main purpose of this thesis was conceived as a comparative study of three strains of carotenogenic yeasts: Rhodotorula glutinis, Sporobolomyces roseus a Cystofilobasidium capitatum. Yeasts were cultivated on different nutrient media, in which selected waste material was used as a nutritional source - rape substrate of two types with different lipid content. For all strains the growth characteristics on rapeseed substrate of the first type with a lower content of lipids were performed. All tested strains were able to use waste substrates as a source of nutrients. Most of strains produced increased amounts of carotenoids, in some cases accompanied with a relatively high production of biomass. As a potentially best producer of enriched biomass yeasts Rhodotorula glutinis and Sporobolomyces roseus were showed. They exhibited the highest production of biomass and metabolites in the media with the ratio glucose:rapeseed cake 3:1 and 1:1. The results showed that rapeseed cake can be used as a potential carbon source for growth of carotenogenic yeasts and production of carotenoids and selected lipids to some limit ratio of waste. Further processing of rapeseeed cake could be a suitable target for following study in related works.
|
guest ::
