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Controlled production of lipids and lipidic substances by selected yeasts and microalgae
Szotkowski, Martin ; Kráčmar, Stanislav (referee) ; Huelva, Ines Garbayo Nores, University (referee) ; Márová, Ivana (advisor)
Karotenoidy jsou přírodní pigmenty vyskytující se v mikroorganismech jako jsou řasy, kvasinky a sinice. Představují nejrozšířenější skupinu antioxidantů s významným biologickým účinkem. V současnosti vzrůstá zájem o karotenoidy vzhledem k jejich příznivým vlivům na lidské zdraví. Chlorofyly jsou zelená fotosyntetická barviva, která nacházejí uplatnění v potravinářství jako intenzivní zelená barviva. Koenzym Q je znám svým pozitivním vlivem pro správnou funkci řady orgánů v lidském těle. Ergosterol je nedílnou součástí membrán kvasinek a hub. Je to provitamin D2, který je důležitou součástí imunitního systému. Mikrobiální lipidy, nebo také ‚‚Single cell oils‘‘ jsou charakteristické vysokým obsahem zdraví prospěšných nenasycených mastných kyselin, které lze využít ve farmacii či kosmetice. Mikrobiální lipidy jsou dále studovány jako alternativa pro výrobu biopaliv. Dizertační práce byla zaměřena na studium a možnosti optimalizace produkce lipidů a lipidických látek vybranými kmeny karotenogenních kvasinek, mikrořas a sinic. V rámci práce byly testované kvasinky rodu Rhodotorula, Rhodosporidium, Cystofilobasidium a Sporidiobolus podrobené kultivacím na sérií médií s různými C/N poměry v rozsahu 13 až 100, obsahujících upravené odpadní substráty z potravinářského průmyslu. Vybrané kmeny byly poté kultivovány v bioreaktorech v médiu obsahujícím kombinaci odpadních substrátů. Kultivace mikrořas rodu Desmodesmus, Scenedesmus, Chlorella, Coccomyxa, Chlamydomonas, Botryococcus se zabývaly optimalizací jednotlivých komponent média a aplikací různých stresů s cílem navýšení produkce studovaných metabolitů. V rámci experimentů s extrémofilní mikrořasou Coccomyxa byly provedeny pilotní velkoobjemové kultivace v otevřených nádržích. V závěrečné části byl provedeny pilotní screeningové a velkoobjemové bioreaktorové experimenty zaměřené na možnosti kokultivace karotenogenních kvasinek a mikrořas. Testované kmeny kvasinek byly s rozdílnou úspěšností schopny utilizovat média obsahující hydrolyzované odpadní substráty. Nejlepším kmenem byl Sporidiobolus pararoseus, který na médiích dosahoval nejvyšších produkcí biomasy i sledovaných metabolitů. Z testovaných odpadních substrátů byla nejlepší kombinace odpadního fritovacího oleje a hydrolyzátu kávové sedliny. Úspěšná optimalizace složení hlavních komponent minerálního média vedla k zvýšené produkci studovaných metabolitů. Největší vliv měl optimální poměr P/N a aplikace oxidačního stresu. Nejlepších výsledků dosáhly mikrořasy rodu Desmodesmus a Scenedesmus. Velkoobjemové kultivace Coccomyxy onubensis potvrdily rezistenci kultury proti kontaminaci vnějšími vlivy a schopnost růstu za vysoké teploty a intenzity světelného záření. Kokultivační experimenty potvrdily schopnost symbiotického růstu kvasinek a mikrořas. Nejlepších výsledků dosahovaly všechny testované kvasinky s mikrořasami rodu Demsodesmus a Scenedesmus a v menší míře i rodu Coccomyxa.
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Brewing industry
Artýszková, Jana ; Illková, Kateřina (referee) ; Omelková, Jiřina (advisor)
The aim of the theoretical part of the thesis is the study and the treatment of the problem of the manufacturing of beer, focused on the czech conditions and classical Czech technology. Within the theoretical part the history of this branch of industry together with the modern methods, enabling greater productivity of beer production, are summarized. Experimental part of the thesis was carried out in a small brewery in Czech republic, producing aproximately 10 000 hl per year. In this brewery the selected parameters were studied not only in the course of cooking and fermentation of beer, but during the output control in racking to the drums and plastic bottles, as well. The studied parameters were as follows: the sugar content of front and wort, the control of microbiological contamination in fermenting cellar and in beer-storage cellar, the content of CO2 by last stage of fermentation and pH- value and color by the end-product.
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Application of microbial oils in cosmetics
Reichertová, Klára ; Uhlířová, Renata (referee) ; Němcová, Andrea (advisor)
This bachelor thesis is focused on the application of microbial oil in a cosmetic product. Eight strains of yeasts of the genus Metschnikowia were examined, namely Metschnikowia pulcherrima 145, Metschnikowia pulcherrima 147, Metschnikowia pulcherrima 149, Metschnikowia andauensis 129, Metschnikowia andauensis 1241, Metschnikowia sinensis 1244, Metschnikowia zizyphicola 1247 and Metschnikowia shanxiensis 1250 for production of microbial oil. Substrates made of technical hemp (hemp flour, hydrolyzate and a mixture of leaves and flowers) were used for cultivation. The cultivation was performed at reduced temperature with the advantage of lipid production. The composition and ratio of fatty acids were determined by gas chromatography According to these resulst was choosen the best lipid producer and the most abundant substrate. This was the strain Metschnikowia zizyphicola 1247 and the substrate of hemp flour. At the end of the practical part was prepared a cream, into which the microbial oil was mixed. Finally, an evaluation of the oil with regard to its use in cosmetics and a comparison with vegetable oil with the same fatty acid profile are given.
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Characterization of selected yeast strains from the environment
Bečková, Alena ; Omelková, Jiřina (referee) ; Vránová, Dana (advisor)
The aim of this bachelor thesis deals with identification type-yeasts, which were obtained from the Culture Collection SAV in Bratislava. The purpose is extension of yeast database in laboratory of Faculty of Chemistry, which is used by identification yeasts in samples taken from food or environment. Identification of yeasts was implemented by method PCR-RFLP. Obtained segment is located in area 5,8S-ITS and is defined by using primers ITS1 and ITS4. Restriction analysis was carried out by restriction endonucleasis HaeIII, HinfI, HhaI and TaqI. Chosen species were also tested for presence of extracellular lipasis. In this part of characterisation was used SpiritBlue Agar. The presence of blue colour is lost by activity of lipasis.
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Study of production properties of Metschnikowia yeasts
Chadimová, Markéta ; Szotkowski, Martin (referee) ; Němcová, Andrea (advisor)
Metschnikowia yeasts are able to produce under different conditions different amounts of lipids, which have remarkable uses in biotechnology and industry. This bachelor thesis is focused on the study of cultivation conditions under which yeasts produce the most lipids and also deals with which fatty acids these triacylglycerols are composed of. The influence of cultivation conditions on the amount of biomass is also monitored. Five yeast strains were examined, namely M. pulcherrima 145, M. pulcherrima 147, M. pulcherrima 149, M. andauensis 129 and M. chrysoperlae 1158. Cultivation media with different C/N ratios, containing glucose or cheap waste substrates (glycerol, coffee and waste fat) were used for cultivation. The cultivations were performed at several temperatures and then the amount of grown biomass was determined, and the composition and ratio of fatty acids were determined by gas chromatography. As yeast that produced the most biomass per time was determined the yeast M. chrysoperlae 1158, which after 336 hours of growth at 15 °C was able to produce 10,66 g/l of biomass on a medium with a ratio of C/N = 100. The highest amount of lipids was obtained with yeast M. andauensis 129 at 8 °C on medium with a ratio of C/N = 100 (21,57 % lipids in dry matter). If biomass production is also taken into account, the best results are obtained by the yeast M. chrysoperlae 1158 at a temperature of 11 ° C on a medium with a ratio of C/N = 100 with a biomass production of 10,15 g/l and 19,58 % lipids in dry matter. This yeast was therefore further cultivated on waste substrates.
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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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Optimalization of PCR-RFLP method for taxonomy of yeasts
Olivová, Radana ; Vadkertiová, Renata (referee) ; Vránová, Dana (advisor)
This thesis deal with optimalization method PCR – RFLP for taxonomy enlistment of yeasts. Conventional identification methods for yeasts are time-consuming. Molecular biological method based on PCR are instrumental towards fast and precise identification as compared to conventional phenotypic methods. In this thesis molecular biological method PCR – RFLP was used for identification and enlistment of yeasts. This metod follow repeating spacers of ribozomal DNA of yeast, characteristic for each species and strain. By the help of PCR were amplified specific partitions of DNA. These fragments of DNA were split by restriction endonucleases and identified by horizontal electroforesis. In background of this thesis there are information about yeasts, their taxonomy and molecular biological methods.
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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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Isolation, Identification and Characterisation of Microbial Communities of Wine and Selected Foods
Šuranská, Hana ; Španová, Alena (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
Proposed dissertation thesis deals with wine and artisanal cheeses microbiology. The first part is focused on identification of yeasts isolated from grapes and musts during production of white and red wines. The grape varieties were grown under the integrated and organic farming on Moravian vineyard. Yeasts were identified by ITS-PCR-RFLP method (amplifying internal transcribed spacer ITS: ITS1, ITS2 and 5.8S rDNA) and unknown species were subjected to partial sequencing of ITS rDNA region. In total, 524 isolates were divided into 14 different species belonging to six genus were identified from. The first stages of fermentation process were characterised by predominance of non-saccharomyces species especially H. uvarum. Due to increased ethanol concentration strains of S. cerevisiae prevailed in the later phases of the process. Further, partial aim of this study was to isolate and to apply selected autochthonous S. cerevisiae strains as starter culture during controlled industrial wine fermentation process. Genus Saccharomyces was distinguished from other non-saccharomyces species by ITS-PCR-RFLP. Further, in order to distinguish Saccharomyces genus at the species and the strain level, several molecular methods were applied including PCR-fingerprinting (rep- and RAPD-PCR), species-specific primers (multiplex and touchdown PCR), LSU-DGGE and interdelta PCR. Species-specific primers enabled us to distinguish some species of the Saccharomyces sensu stricto complex. Furthermore, interdelta PCR seems to be useful tool for S. cerevisiae strains identification. Among 120 isolated autochthonous strains belonging to Saccharomyces genus, 45 different strains were identified. Based on its sufficient technological properties (osmo- and ethanol tolerance, low H2S production etc.), S. cerevisiae 1-09 strain isolated from grape berries coming from moravian vineyard was chosen. Strain S. cerevisiae 1-09 was tested in small amount of must and after that also during industrial fermentation of red and white wine production. Based on the results of chemical and sensorial analysis, the strain seems to be suitable for application as the starter culture for winemaking process. The final part of this thesis is focused on quantification and identification of the yeasts isolated from artisanal cheeses and their by-products coming from Western Balkan Countries. Isolated species were identified by ITS-PCR-RFLP, partial sequencing and by physiological tests. Among the 20 yeast species found, D. hansenii, C. zeylanoides and Y. lipolytica were found to be predominant. Moreover, we developed culture-independent, semi-quantitative technique based on construction of ITS-clone library from metagenomic DNA to investigate complex fungal communities associated with artisanal cheeses and their by-products. Novel technique is based on direct extraction of total DNA from the sample. This was compared with culture-dependent ITS-PCR-RFLP and culture-independent LSU-DGGE methods. The results highlighted the discrepancies among these methods. Finally, the divergences among applied methods were confirmed by correlation analysis and by indices of general biodiversity and dominance of species. ITS-clone library approach combines the advantages of cultivation-based analysis and LSU-DGGE with semi-quantification of fungal species without the requirement of their cultivation. This study might open new perspectives in direct and complex analysis of yeasts and moulds in food matrices.
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Identification of yeasts from interspecific varieties of grapes
Sadel, Peter ; Omelková, Jiřina (referee) ; Vránová, Dana (advisor)
The main goal of my diploma thesis was to identify and characterize yeasts from must Hibernal and also collection yeasts by using methods called RFLP-PCR (Restriction Fragment Length Polymorphism - Polymerase Chain Reaction). Theory was the first part of my diploma thesis which dealt with wine, yeasts and molecular methods. Theory section was followed by experimental section divided into two parts. The main goal of the first part was to characterize and identify yeasts from must Hibernal by using PCR and RFLP-PCR methods. In the samples there were found yeasts Saccharomyces and Pichia. The second experimental part of my diploma thesis had a goal to extend the database of new yeasts using the same methods mentioned in the first part of experimental section.
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