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Life cycle of Haematococcus pluvialis (Chlorophyceae) species complex
Vávrová, Karolína ; Němcová, Yvonne (advisor) ; Nedbalová, Linda (referee)
Freshwater green algae within species complex Haematococcus pluvialis are best natural producers of secondary carotenoid astaxanthin famous for its strong antioxidant activity. Despite worldwide distribution, great biotechnological potential and extensive literature dealing with different approaches to the cultivation of these organisms, there still remains surprising gaps in knowledge of their complex life cycle and behaviour. First uncertainty concerns asexual cell division of zoospores, hypothetically limited to five cell doublings (Lee & Ding 1994). No such genetic limitation was observed in this thesis, number of cell divisions is thus influenced mainly by cultivation conditions. There is still considerable uncertainty regarding to the asexual reproduction, as well. Ambiguity might be caused by scarce observation of the whole process of gametogenesis and conjugation of gametes. Published studies differed in some particular points. Unfortunately, we did not succeed in induction of gametogenesis despite broad scale of tested cultivation conditions applied to two different species within the H. pluvialis complex, namely H. pluvialis and H. rubicundus. This failure might be caused by heterothalism of the studied strains. Haematococcus pluvialis inhabits mainly ephemeral pools. Its ability to...
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Cell cycle of astaxanthin producing green alga Haematococcus pluvialis, culture media and cultivation conditions
Vávrová, Karolína ; Němcová, Yvonne (advisor) ; Pichrtová, Martina (referee)
Haematococcus pluvialis is unicellular green alga (Chlorophyceae, Chlamydomonadales), which can be found in ephemeral rock pools and birdbaths. It is the best natural producer of strong antioxidant astaxanthin, red secondary carotenoid used as coloring agent in aquaculture and poultry breeding. Astaxanthin also has various positive effects on human health. Haematococcus pluvialis has quite complex life cycle consisting of four life stages, biflagellated zoospores, non- motile round palmella stage, thick-walled akinetes (aplanospores, cysts) with high content of astaxanthin and small biflagellated gametes. Akinetes, which enable H. pluvialis to survive desiccation in its natural habitat, are formed in response to stress conditions such as high irradiance and temperature and nutrient depletion. Cells undergo dramatic ultrastructural changes during aplanospore formation. Chloroplast volume is decreased, synthesis of high amount of astaxanthin and fatty acids results in presence of lipid droplets that spread from the center to the periphery of the cell and thick cell wall containing algaenans is formed. Cultivation of H. pluvialis is often performed in two subsequent stages, green stage focused on biomass production and red stage focused on astaxanthin accumulation under stress conditions. There are...
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Diversity, ecology and ecophysiology of snow algae
Mikešová, Lenka ; Nedbalová, Linda (advisor) ; Kvíderová, Jana (referee)
Permanent and temporary snow cover in polar and mountain areas is a very extreme habitat. However, there are organisms that inhabit this environment. Green algae of the order Chlamydomonadales (Chlorophyta) belong among the important representatives of cryoseston, which are known from polar and mountain regions around the world. These organisms, which occupied the snow environment probably secondarily, developed different ecophysiological adaptations required for the successful survival in extreme conditions. Low temperatures and high levels of radiation are the main extreme factors that organisms must cope with. The most important survival mechanisms include the adaptation of life cycles (such as of the shifts between resistant stages and moving vegetative stages), accumulation of secondary carotenoids, changes in fatty acid composition of membranes and lowering of growth temperature optima in some species. These adaptations together with the study of diversity represent at present the main subjects of research of snow algae.
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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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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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Miniaturized techniques for analysis of industrial yeasts
Obalil, Jiří ; Kočí, Radka (referee) ; Klepárník,, Karel (advisor)
Carotenoids are natural pigments that have antioxidation and antimutagenic abilities. They are produced with the help of new technological methods. For example, carotenoid yeast Rhodotorula glutinis produces -carotene with the yield of up to 6 – 10 mg/g of the dry substance. The method of the mass spectrometry with the nanoelectrospray in the positive mode was optimized for the determination of -carotene, lycopene and astaxanthin in this project. Ionizing voltage of 4 kV and the sample flow rate of 15 – 80 nl/min through the spray silica fused capillary with the internal diameter of 25 µm were found to be the optimum parameters of the analysis. A mixture of chloroform with the addition of ammonia was used as a spray solvent for both standard and cellular samples. During the process of ionization by nanoelectrospray, -carotene and lycopene form cation radical [M] • + with the molecular mass to charge ratio (m/z) of 536, while asthaxanthin forms the protonated molecule [M + H]+ with the m/z of 597. The partial lysis of individual Rhodotorula glutinis cells was demonstrated under microscope in the organic solvents tetrahydrofuran and dimethylsulfoxide. Chloroform, acetone, acetonitrille, methanol and isopropanol did not affect the cells after a 15 min treatment.
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Metabolite production by some strains of industrial yeasts in various phases of cell growth
Jankeje, Kristína ; Kubešová,, Jitka (referee) ; Kočí, Radka (advisor)
Presented bachelor thesis is focused on industrial application of chosen yeast strains. Principal interest of work is to study production of primary and secondary metabolites during individual growth phases. Optimal growth conditions as well as influence of exogenous stress factors (mainly oxidative and/or salt stress) on cell growth and yeast metabolism are discussed. In experimental part growth curve of industrial strain Phaffia rhodozyma was determined. Biomass increase (maximum in 90th hour 5,441 g/l), astaxanthin production (secondary metabolite) and/or ergosterol biosynthesis (primary metabolite) were observed. The best ration of astaxanthin to total carotenoids was 50 %. Next studied metabolite was ergosterol, its total amount in dry biomass was 0.11 %. In conclusion astaxanthin amounts produced in optimal growth conditions were compared with yields obtained under stress cultivations. Results of stress experiments illustrate positive influence of stress factors on cell growth as well as on astaxanthin biosynthesis. Low concentration of salt (2% NaCl) added in inoculum with 5 mM hydrogen peroxide in production medium would be the best combination in industrial applications.
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