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Use of autotrophic algae to production of biomass and some metabolites
Chrástová, Nikola ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
Chlorophyll and carotenoids are natural pigments and antioxidants. These pigments are suitable for use in the pharmaceutical, cosmetics or food industries. Lipids are another major metabolite of algae and are a potential feedstock for biofuel production. This bachelor thesis deals with the cultivation of selected genera of autotrophic algae, subsequent isolation of these selected metabolites and their application. The theoretical part focuses on the description of the algae, the metabolites produced, the cultivation and the chosen methods. The experimental part deals with the optimization of the cultivation of algae of the genera Chlorella, Chlamydomonas and Desmodesmus, depending on the chosen medium and intensity of illumination, determination and quantification of algal biomass. Further, extraction of lipidic and carotenoid substances and determination by HPLC and GC was performed. In the overall comparison, the best producer of biomass is the algal genus Desmodesmus and the highest production of pigments and lipid substances was reached in algae of the genus Chlamydomonas.
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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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Potential use of waste lipid substrates and glycerol to production of carotenoids by yeasts
Kostovová, Iveta ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The aim of this study was use of lipid waste substrates and waste glycerol as carbon sources for red yeasts. Six yeast strains, namely Rhodotorula glutinis, Rhodotorula rubra, Rhodotorula aurantiaca, Cystofilobasidium capitatum, Sporobolomyces roseus and Sporobolomyces shibatanus were used for cultivation. All studied yeast strains were able to utilize glycerol and rapeseed oil as a carbon source. Cultivation in oil medium was in most of strains accompanied by decreased production of carotenoids. Increased production of ergosterol, lycopene and biomass was observed in some strains only. Most of yeast strains cultivated in glycerol medium produced increased amount of biomass as well as carotenoids. The best producer of bi-omass and pigments was yeast strain Sporobolomyces roseus CCY 19-6-4 grown in medium containing glucose and glycerol in a 1:1 ratio.
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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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Characterization of yeast and algae metabolites by instrumental techniques
Kurowska, Klára ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
The presented diploma thesis deals with the analysis and production of selected microbial metabolites of the yeast Rhodotorula kratochvilovae in comparison to various species of microalgae. The theoretical part summarizes the knowledge about the conditions for optimal growth and production of secondary metabolites. Analytical methods, by which metabolites were quantified and evaluated, were also described. In the experimental part of this thesis, the amount of accumulated biomass, exoglycolipids, intracellular lipids, phycobiliproteins and carotenoids according to various nutrient sources in the culture medium was studied. Specifically, the yields of these metabolites were investigated in the utilization of various sources of nitrogen (yeast extract, urea, ammonium sulfate, ammonium chloride and potassium nitrate), carbon (glucose, mannose, xylose, glycerol and lactose) and at variable C / N ratios (20; 40; 70; 100 and 120: 1) ideally selected sources of observed nutrients in partial experiments. The last part of the experiment was focused on comparison of the analysis of metabolites of autotrophic algae (chlorophylls, phycobiliproteins and lipids).
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Cultivation of microalgae and carotenogenic yeasts under stress conditions
Sniegoňová, Pavlína ; Skoumalová, Petra (referee) ; Szotkowski, Martin (advisor)
In biotechnological industry we can register greater use of microorganisms like microalgae and carotenogenic yeast for production of selected substances or for valorisation of waste materials. Among metabolites produced by these microorganisms are carotenoids, which are natural pigments with antioxidant and other biological effects. Other significant produced substances are lipids, which represent large range of substances in living organisms. They form cellular membranes and serve as a reservoir of energy. These substances can be used in food, cosmetic and pharmaceutical industry and also in production of biofuels. Theoretical part is focused on description of selected genera of microorganisms, their cultivation and produced metabolites. Stress factors to which they were exposed are also mentioned, as are the analytical methods used for detection of produced metabolites. Next part is dedicated to the comparison of production properties of selected microorganisms depending on used stress factor. Increased production of lipids in yeast is observed in C. macerans and S. metroseus with higher concentrations of FeCl3. While using whey as a source of ccarbon, yeasts show decreased production of carotenoids. Highest production of lipids in algae and cyanobacteria was observed in S. acutus and D. quadricaudea. Production of carotenoids depended on applien stress factor and highest productions was observed in C. sorokiniana, C. reinhardtii, D quadricaudea and Coccomyxa sp.
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Study of red yeast metabolism on molecular level
Roubalová, Monika ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.
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Production of lipolytic enzymes by yeasts
Bradáčová, Kristína ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on controlled production of lipolytic enzymes, bioactive substances and lipids by carotenogenic yeasts. Theoretical part deals with characterization of lipolytic enzymes, carotenoids, lipids and their properties, possibility of production and application. In experimental part the enzymes, carotenoids and lipids were produced by red yeasts Rhodotorula mucilaginosa, Cystofilobasidium macerans and Sporidiobolus salmonicolor by submerged cultivation in mineral medium with different additions: glucose, glycerol, fat, fat with glucose, fat with polysorbate 80, fat with glycerol, fat with polyethylene glycol, fat with higher and lower addition of palmitic acid, enzymatic fat hydrolysate, acidic hydrolysate a basic hydrolysate. The activity of extracellular lipase was monitored in medium after 96-hour cultivation. Concentration of -carotene, total carotenoids, ergosterol and ubiquinone was determined by HPLC, concentration of fatty acids and amount of fat by GC. Production had differed depending on used yeasts and substrate. As the best producer of carotenoids Cystofilobasidium macerans was found, ergosterol was highly produced by Sporidiobolus salmonicolor. The production of ubiquinone was almost equivalent in all yeasts and lipolytic activity was the highest in Sporidiobolus salmonicolor. The patricular medium sample with high lipolytic activity was further separated and analysed by ultrafiltration and PAGE-SDS electrophoresis. This diploma thesis was done within the international project ,,LipoFungi“.
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Valorization of waste animal fat by carotenognic yeasts
Chrástová, Nikola ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with valorization of waste animal fat using carotenogenic yeast and conversion to biomass enriched with valuable substances. These microorganisms have the ability to utilize various waste substrates and convert them into interesting products such as carotenoids, lipids, ergosterol and ubiquinone. Four strains of yeast (Rhodotorula glutinis, Cystofilobasidium macerans, Rhodotorula mucilaginosa, Sporidiobolus pararoseus) were used. The theoretical part is focused on characterization of yeasts, enzymes and lipases, produced metabolites, yeast and description of analytical methods. In the experimental part, cultures were performed at four C/N ratios on media with different carbon sources, which were glucose, glycerol, fat, fat with added emulsifier, enzymatically hydrolyzed fat and fat with enzyme addition. Biomass was determined gravimetrically for all samples. Carotenoids, ergosterol and ubiquinone were analyzed on an HPLC/PDA equipment. The intracellular lipid content was determined by GC/FID. The production of these substances was different in individual strains depending on the carbon source and the C/N ratio. Rhodotorula glutinis is the most suitable for producing biomass and carotenoids. The highest amounts of ergosterol and ubiquinone were produced by Cystofilobasidium macerans. Sporidiobolus pararoseus had the largest lipid content in biomass. This diploma thesis was elaborated within the international project "LipoFungi".
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Optimization of extraction of metabolites produced by selected strains of microalgae and carotenogenic yeast.
Obračaj, Jan ; Holub, Jiří (referee) ; Szotkowski, Martin (advisor)
This bachelor thesis is focused on the production of microbial biomass using selected strains of cyanobacteria and carotenogenic yeasts. Subsequent preparation of extracts and study of their stability under different temperature conditions with the addition of the antioxidant tocopherol. In the experiment, 2 series of extracts with hexane and ethanol were prepared. The theoretical part of the work describes the studied metabolites carotenoids, chlorophylls, ubiquinone, ergosterol and microbial lipids. Furthermore, the used strains of cyanobacteria and carotenogenic yeasts are described here. Finally, methods for extraction and analysis of metabolites are described. The experimental part describes the used cultivation techniques, preparation of microbial extracts and used analytical methods. The results part of the work then contains a summary of the results of long-term stability tests of extracts. The results of the work clearly confirm the temperature dependence of the stability of individual metabolites. This phenomenon was most evident in chlorophyll and carotenoid pigments. Hexane appears to be the best solvent in terms of the stability of the substances used. The results of this work provide important information in terms of the economics of storage of these valuable substances usable in the food, pharmaceutical or feed industries.
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