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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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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Use of Waste Substrates to Production of Enriched Yeast Biomass
Starečková, Terezie ; Demnerová, Kateřina (referee) ; Vávrová, Milada (referee) ; doc.PharmDr.Petr Babula, Ph.D. (referee) ; Márová, Ivana (advisor)
Yeasts are like other organisms constantly exposed to environmental influences. Their survival depends on the skills to adapt to environmental changes, including the ability to use various alternative sources of nutrients. In presented PhD thesis carotenogenic yeast belonging to the genera Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates, and also subjected to several types of exogenous stress effects and mutations in order to increase the production of microbial biomass enriched with specific metabolites. As alternative nutrient sources derived from waste substrates from agricultural and farm production apple peel, pulp, corn germ and more were tested. Yeasts were also exposed to osmotic, oxidative and combined stress (benefits of various concentrations of NaCl and H2O2 to the culture media), followed by metal ions of selenium and chromium in concentrations of 0.01 mM, 0.1 mM and 1 mM. The effect of mutagen methanesulfonic acid ethyl ester was tested too. In all experiments the adaptivity of cells, morphological changes, color pigments produced by the media while some important fungal metabolites production and changes in chromosomal DNA fragmentation were analyzed. In order to evaluate potential changes in the yeast genome after treatment with mutagen and stress factors methods for isolation of intact chromosomal DNA and DNA analysis by pulsed field gel electrophoresis was optimized. The amount of produced metabolites was mainly analyzed by RP-HPLC with UV/VIS and MS detection. The work has been shown that most strains are able to use waste substrates and produced selected target metabolites. Biomass, for example, in R. aurantiaca on apple fiber was about 7 g/l and in C. capitatum cultivated on modified whey reached to 9 g/l. Amount of produced carotenoids by R. aurantiaca cultivated on wheat germ and maize after enzymatic hydrolysis by F. solani was 1.01 mg/g and S. roseus on pasta 4.3 mg/g. The values of ergosterol synthesis in R. aurantiaca are on the apple shells around 4.8 mg/g, in S. roseus on pasta with the enzymatic hydrolysis of P. chrysosporium 8.9 mg/g. The best substrate for biomass production and induction of carotenoids are waste substartes containing a mixture of simple and complex carbohydrates enriched with the addition of nitrogen compounds. Potential cytotoxic effect of stress factors of low concentrations was demonstrated. Red yeast genome was able to distribute by optimized PFGE, the karyotype of tested yeasts contain 11 or more chromosomes with visible differences between yeast species and genera. During exchange internship the ability of recombinant yeast S. cerevisiae to convert xylose to xylitol, which would be achieved by increasing the production of bioethanol as alternative fuel sources was studied. It turned out that both ligninocellulose materials to bioethanol production, as well as various waste substrates for microbial synthesis of carotenoids would reduce costs for industrial production of yeast metabolites, as well as to reduce the negative burden on the environment.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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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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Encapsulation of natural antioxidants
Štindlová, Jitka ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
In theoretical part of this diploma thesis the basic properties of antioxidants, especially anthocyanins and phenolic compounds are described, as well as the basic features and principles of free radicals formation. The theoretical part also describes some possible ways of encapsulation of antioxidants into polysaccharide and lipid particles. In the experimental part basic characteristics of extracts from selected lyophilized fruits and vegetables (carrots, apples and mixed berries) are described. As group parameters of plant extracts the total antioxidant activity, content of flavonoids and phenolics, carotenoids, anthocyanins and ascorbic acid were determined. In experimental part also various encapsulation techniques were tested, encapsulation effectiveness of each technique was evaluated and the stability and size of the created particles were determined. As the best encapsulation method in terms of encapsulation efficiency in most of samples/parameters ethanol injection was found. On the other hand the particles prepared by ethanol injection are relatively unstable in terms of zeta potential, which is followed by their tendency to aggregate. As the most stable particles prepared by thin layer evaporation (TLE) and reverse phase thin layer evaporation (RP-TLE) were evaluated. Particles prepared by TLE, RP-TLE and chitosan-alginate particles exhibited a negative charge, while particles prepared by ethanol injection stayed uncharged and chitosan particles have a positive charge.
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Production of carotenoi by yeasts of the genus Cystofilobasidium
Vavrysová, Alena ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
Carotenoids are important industrial pigments present practically in all living organisms. The aim of presented work is the study of regulation of carotenoid production in yeasts of the genus Cystofilobasidium in presence of exogenous stress factors. Growth curve of C. capitatum exhibited typical two-stage course with prolonged stationary phase similar to other carotenogenic yeasts. Maximal production of biomass and beta-carotene occurred in 103rd hour. Applied stress factors (2-5% NACl, 2-5 mM H2O2, 0,01-1 mM Se(IV), 0,1-5 mM Cr(III)) exhibited no significant influence on biomass production, which reached on average 8-9 g/l. Positive effect was observed in presence of 5mM Cr where 10 g/L of biomass was produced. Beta-carotene formation was positively influenced by many applied stress factors, the highest yield (695 g/g) was reached in presence of 0,1 mM Se(IV). No simultaneous regulation of ergosterol and carotenes was observed in Cystofilobasidium cells. Production properties of yeast strain C. capitatum CCY 10-1-1 wee compared with those of other carotenogenic yeasts of the genes Rhodotorula and Sporobolomyces. C. capitatum produced similar biomass yield as Rhodotorula sp. in presence of salt. Production of beta-carotene by C. capitatum was slightly higher than in Rhodotorula glutinis, but lower than in Sporobolomyces strains which exhibited substantially lower biomass production. Karyotype of C. capitatum is relatively different when compared with karyotype of other carotenogenic yeasts. Based on summary of our results in seems that yeasts C. capitatum exhibit similar physiological as well as production properties as some Rhodotorula strains. Thus, yeasts of the genus Cystofilobasidium could be potentially used to industrial production of carotenoid pigments as well as yeast biomass rich in carotenoids and some biogenic elements.
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