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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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Study on PHA accumulation by cyanobacteria
Pacasová, Viktorie-Alexandra ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis was focused on studying the influence of selected stress factors on PHB production in strains of cyanobacteria Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Cyanobacterial stains were cultivated in multicultivator and as a stress factor an increased salt (sodium chloride) as well as a mixture of acetate and -butyrolactone was selected. The PHB content in cyanobacteria was determined by gas chromatography. In addition, the content and presence of pigments in cyanobacteria strains was determined by pigment extraction as well as using direct spectroscopy of whole cells. When cultivated with an increased salinity, the hypothesis of an increased PHB production was not confirmed most likely due to and overstressing the Synechocystis salina CCALA 192 cultures with increased salt content, constant illumination of multicultivator and by nitrogen limitation due to the used medium. When cultivated with the mixture of acetate and -butyrolactone the stains were analysed in the same way as in the cultivation with an increased salinity. In this case, the presence of 3-hydroxybutyrate and 4-hydroxybutyrate copolymer was determined in the stain cultivated with the mixture of acetate and -butyrolactone. During this cultivation, a positive effect of initial concentration of cells in the cyanobacterial cultures combined with continuous illumination and the type of nitrogen-limiting type of medium on the growth rate of selected stains was observed.
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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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Use of algae extracts in cosmetics
Plášková, Anna ; Rapta, Marek (referee) ; Márová, Ivana (advisor)
This thesis is focused on a preparation of extracts from selected species of algal and cyanobacterial strains, their characterization, a cytotoxicity testing and a use in cosmetic products. The theoretical part contains the review of a taxonomic classification and parameters of algae and cyanobacteria and basic information about used organisms. There is a description of the most significant factors influencing the algae cultivation and production of biomass. Further, active substances of algae and cyanobacteria, which could be used in cosmetic products as UV protective substances, were described. An overview of extraction techniques, encapsulation of active substance into liposomes and particles characterization, antioxidant activity analysis, and cytotoxicity tests is given too. In experimental part, antioxidant activity of algal extracts and chlorophyll a and carotenoid content were determined spectrophotometrically. The long-term stability of extracts was monitored for three weeks. Liposomes were prepared by sonification and encapsulation activity was analysed. The Dynamic Light Scattering method (DLS) was used for analysis of polydispersity and size of liposomes. Colloid stability of particles was determined by zeta potential. Cytotoxicity tests were performed using MTT assay. The prepared extracts, particles and lyophilized biomass of algal and cyanobacterial strain were further used as active substances in the formulation of cosmetic products (type o/w). These creams were tested on human skin.
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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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Application of chemical and physical stresses in the late phase of growth to selected strains of microorganisms
Langer, Marek ; Holub, Jiří (referee) ; Szotkowski, Martin (advisor)
A wide spectrum of microorganisms is used in the biotechnological industry for production of meaningful metabolites; including carotenoids and chlorofyles with antioxidant effects, fatty acids, ubiquinone and ergosterol. Ways to increase yield of metabolites to maximum capacity are being researched to increase efficiency and economical sustainability in industry. One of the ways is the application of chemical stress factors on microorganisms as is described in this thesis. In the theoritical part certain microorganisms, their important intracellular metabolites and their metabolism are established. The last subchapter covers the usage of stress factors in industry. In the experimental part each microorganism was subjected to various concentrations of sodium chloride, cobalt(II) sulfate and iron(III) chloride. Significant increase of lipid production was registered after an application of sodium chloride on yeast strain Rhodosporium toruloides. An increase in production of torularhodin in Sporidiobolus pararoseus occured after application of iron salt. After an application of sodium chloride the growth of torularhodin in strains Cystofilobasidium macerans a Rhodosporidium toruloides was significant. Sodium chloride was also a suitable stress factor for lycopene formation in Rhodotorula mucilaginosa. The most meaningful stress factor of algae was cobalt(II) sulfate which increased metabolite production in Scenedesmus obliquus. Cyanobacteria Anabaena torulosa also attained a significant production in the presence of sodium chloride.
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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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Evolutionary engineering of cyanobacteria with respect to PHA accumulation
Vašířová, Kristýna ; Slaninová, Eva (referee) ; Samek, Ota (advisor)
The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.
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Preparation of organic fibers with the addition of algae extracts
Tuhrinská, Terézia ; Skoumalová, Petra (referee) ; Němcová, Andrea (advisor)
The presented bachelor thesis is focused on the preparation and characterization of organic fibers with an admixture of pre-prepared extracts of microalgae and cyanobacteria containing valuable active compounds. The theoretical part describes selected metabolites of microalgae and cyanobacteria, methods of their analysis, the issue of encapsulation and incorporation of active substances into nanofibers and presents some methods of their preparation. In the experimental part of this thesis, selected strains from the CCALA collection were first cultured. Subsequently, aqueous, ethanol and hexane extracts of biomass from cultured and commercial microalgae and cyanobacteria were prepared. The prepared extracts were spectrophotometrically evaluated in terms of the concentration of chlorophylls, carotenoids, antioxidants, polyphenols, and total proteins present. In addition, 96 % ethanol extracts were subjected to more accurate analysis of chlorophylls and carotenoids by HPLC. The highest antioxidant activity was observed for aqueous extracts. Gelatin nanofibers containing selected extracts were formed by electrospinning method. The formed fibers were examined afterwards to identify a mass fraction of incorporated microalgal and cyanobacterial substances. The fibers were further tested for antioxidant activity with the TEAC assay. The most pronounced antioxidant effect was detected for the fiber with incorporated aqueous extract of the cyanobacterium Arthrospira maxima. Finally, the fibers were tested for their safety in contact with human cells. The amount of fibers used did not show any cytotoxic effect on human keratinocytes and the tested materials can thus be considered safe for application in cosmetics.
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Spectrophotometric determination of phosphate in ternary systems with dyes
Novotný, Radek ; Komendová, Renata (referee) ; Sommer, Lumír (advisor)
The theoretical part of this master’s thesis is focused on the characteristics and forms of phosphorus, occurrence in natural waters and the problems associated with higher phosphorus concentrations, especially in the development of cyanobacteria and algae. There are also described the possibilities of determining the degree of pollution of natural waters by nutrients and the effects of eutrophication. There are also described methods for the determination of phosphates, especially spectrophotometric determination in ternary systems with dyes. Like other methods are mentioned AAS, ion chromatography, electrophoresis and izotachophoresis, electrochemical methods or enzymatic methods. In the experimental part of this work deals with the selection and optimalization of suitable ternary system with organic dyes for spectrophotometric determination of orthophosphates. The system with phosphovanadomolybdic acid is studied too. For analytical procedure with malachite green oxalate, which was found to be most useful, were studied interfering ions, commonly occurring in natural waters . The chosen procedure was finally tested on real water samples.
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