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Metabolism of Bacterial Cells and the Effect of Stress on Biosynthesis of PHA
Kučera, Dan ; Kráčmar, Stanislav (referee) ; Ondrejovič,, Miroslav (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of polyhydroxyalkanoate biosynthesis as a microbial product with the potential to replace current conventional plastics made from petroleum. The dissertation thesis is elaborated in the form of a discussed set of already published publications, which are then part of the thesis in the form of appendices. The work builds on relatively extensive knowledge in the field of polyhydroxyalkanoate production and brings new facts and possible strategies. Various possibilities of analysis of polyhydroxyalkanoates using modern methods were tested in this work, which brings especially speed, which can be crucial in real-time evaluation of production biotechnological process. Raman spectroscopy has proven to be a very promising technique for rapid quantification of PHA. Furthermore, the work deals with valorisation of waste of food and agricultural origin. Emphasis is placed on methods of detoxification of lignocellulose hydrolysates. In this context, adsorption of inhibitors to lignin was first used as an alternative to other detoxification techniques. Due to detoxification, selected production strains Burkholederia cepacia and B. sacchari were able to utilize softwood hydrolyzate for PHA production. In the next part of the work was also tested the possibility of using chicken feathers as a complex source of nitrogen. Evolutionary engineering was also used as a possible strategy to eliminate the inhibitory effect of levulic acid as a microbial inhibitor that results from the hydrolysis of lignocellulosic materials. Adaptation experiments were used to develop strains exhibiting higher resistance to levulic acid and the ability to accumulate a higher 3HV copolymer from the original wild-type C. necator strain. Another promising approach tested in the work was the use of extremophilic microbial strain, which leads to a reduction in the cost of biotechnological production. Selected Halomonas species have shown high potential as halophilic PHA producers. The final part of the thesis was devoted to the selection of the production strain with regard to the properties of the resulting PHA. The Cupriavidus malaysiensis strain was selected to produce a P(3HB-co-3HV-co-4HB) terpolymer which revealed significant differences in material properties over P3HB.
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Physicochemical Aspects of Ecological Heat-transfer Fluids
Skolil, Jan ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (referee) ; Čorňák, Štefan (referee) ; Čáslavský, Josef (advisor)
This thesis develops composition of heat transfer fluids with antifreeze properties next to traditional one component glycols produced from propene and ethene. Based on assessment of physical properties it considers application of progressive antifreeze compounds of glycerine and propan-1,3-diole and their mixtures with classic glycols, which do not have negative influence on the environment and which are produced in last years from renewable sources. Due to the necessity of selection of certain compromise between their heat transfer properties, price, solubility and other factors physical properties of selected mixtures are determined together with synergy of each compound, including formulas with glycols: ethan 1,2 diol and propan 1,2 diol Another evaluated factor of studied formulations in addition to their heat transfer properties was anticorrosive protection of common corrosion inhibitors mixtures. The result of whole work is the proposal of several most perspective formulations based on mixtures of polyhydric alcohols in terms of their features enabling them heat transfer, reasonable costs, and the restrictions of impact of their use, particularly in heat transfer systems, to human health and the environment. The knowledge of physical properties of water solutions of polyhydric alcohols mixtures also helps to better using of recycled materials from planning recycling plant for glycols.
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Study of Exposure PBDE and Perfluorinated Compounds into Aquatic Ecosystem
Vondráčková, Ilona ; Chýlková, Jaromíra (referee) ; Hroch, Martin (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
In the presented PhD. thesis, problems covering occurrence of polybrominated diphenyl ethers (PBDE) and perfluorinated compounds in the environment were solved. The study was focused on identification and further verification of optimal methods in order to determine PBDEs and perfluorinated compounds sampled from the aquatic ecosystem. Polybrominated diphenyl ethers belong to persistent compounds classified as main pollutants; within the environmental constituents, they have been observed particularly in the last decade. For these studies, there were selected surface water matrices and sediments taken in various localities within the river Svratka basin. The analyses were to demonstrate whether they accumulate and remain in these specific matrices for longer time. There we assessed the congeners of polybrominated diphenyl ethers as follows: BDE-28, 47, 66, 85, 99, 100, 153, 154 and 183. In order to isolate them from matrices, various extraction techniques were used, i.e., ultrasonic extraction, microwave extraction, and pressure solvent extraction. Gas chromatography method with electron capture detection (GC/ECD) was selected for determination. The accomplished studies also assessed basic chemical, physical and environmental characteristics of diphenyl ethers in the environmental constituents. The attention was also paid to perfluorooctanoic compounds (PFOA), (PFOS) and (FOSA); physico-chemical properties of these organic pollutants were characterised and their toxicological and environmental aspects were evaluated. Usability of extraction techniques (ultrasonic extraction, pressure solvent extraction, solid phase extraction) applied to PFCs isolation from sediments samples was assessed. Identification and quantification of these analytes were performed using a high performance liquid chromatography/mass spectrometry method (HPLC/MS). After that, the optimized methods applied to real samples. Surface water and sediments were sampled within the river Svratka basin, in particular, from 19 sampling localities. PBDE occurrence was confirmed in sediments samples from the Svratka river (g.kg-1), PBDE were not detected in the surface water samples, their concentrations were below the detection limit, resp.; perfluorinated compounds were not detected in sediments and water samples at all.
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The Study of Distribution of Halogenated Diphenylethers to Parts of Environment
Hroch, Martin ; Hajšlová, Jana (referee) ; Kráčmar, Stanislav (referee) ; Šucman, Emanuel (referee) ; Vávrová, Milada (advisor)
In recent years considerable attention is paid to problems of brominated flame retardants (BFR's – Brominated Flame retardants), which are a diverse group of organic compounds. Even in the recent past have been the most often represented group particularly polybrominated diphenyl ethers (PBDEs), which have found wide use in many industrial sectors. The reason of aplication of these substances is the ability to slow down the combustion process and reduce the risk of ignition by the excessive heating of material. On the other hand, serious concerns about the use of PBDEs was added. The most serious trouble is particular their possible to release from consumer products during their normal use, toxicity and high persistence in parts of environment. Some of these contaminants are more characterized by a high degree of accumulation in biological systems. In this work by several sub-studies the issue of assessment the level of pollution of the aquatic ecosystem of selected localities of the Czech Republic just PBDEs was addressed. One of objectives was monitored the situation of breeding ponds near the village Záhlinice in Central Moravia. Here was assessed level of contamination in fish and birds as two consecutive segments of food chain. The obtained results largely confirmed the assumption that with increasing trophic level of organisms leads to bioaccumulation and thus also higher findings. For comparison, the capture of fish and birds in other localities of the CR was also implemented. In Bartošovice and Hustopeče nad Becvou levels at the buzzard and heron were detected as comparable. The cormorants of the order higher levels of PBDEs were found. Further were also evaluated levels of PBDEs detected in individuals of bioindicator kind of chub caught in two locations on the river Svratka in spring and autumn. Findings of PBDE in muscle, skin and intestines were compared. The obtained results show that between tissues and sites wasn’t significant variability respectively and the findings of controlled substances are practically comparable. With aim to determine the dependence of the concentration of PBDEs in organisms on the length of the river was conducted monitoring of Vír and Brno water reservoirs. In both locations was main monitored kind of bream, which was completed by the other species. The results of the analysis indicate a higher total concentration at the lower part of river Svratka. Differences were also the distribution of individual congeners in both sampling locations. For monitoring of terrestrial system contamination levels of PBDEs has been selected several types of plant bioindicators. These are mainly pine needles (Scots pine, Eastern white pine, Silver fir, Blue spruce) collected from different localities of the Czech Republic. Other matrices were chosen roughages (Common wheat, Naked barley, Alfalfa wheat, Red clover) and oilseeds (Rapeseed) from the Novy Jicin, where Agriculture plant school of the University of Veterinary and Pharmaceutical Sciences Brno. Detected values of PBDE concentrations were very low, generally ranged at the detection limits of the analytical method.
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Preconcentration Techniques for Determination of Uranium Using the Modified Sorbents.
Moos, Martin ; Řehůřková, Irena (referee) ; Kanický, Viktor (referee) ; Kráčmar, Stanislav (referee) ; Sommer, Lumír (advisor)
The work has been focused on the preconcentration techniques for determination of uranium on the modified sorbent, with the final use of ICP-OES and ICP-MS. First ICP-OES at a wavelength of 385.958 nm was used for determination of uranium. Parameters were optimized and the effect of mineral acids (HCl, HNO3), tensides (Septonex, Ajatin) and organic reagents like (4-(2-pyridylazo)resorcinol, ammonium pyrrolidinedithiocarbamate, 8-hydroxyquinoline-5-sulphonic acid and 1,2-dihydroxyanthraquinone-3-sulphonic acid) was observed. For ICP-MS an isotope 238U was chosen. As an internal standard 209Bi (200 ?g•l-1) was used. Modified silica gel was used (Silicagel tethered with C18, C8 alkyls and phenyl) and two types of Amberlite XAD 4 and XAD 16 for the preconcentration of uranium (VI). Commercially produced Amberlite was modified as follows: Drying at 100 °C (24 hours), milled, minced sorbent was selected and fractionated to particles size ranging from 0.32 to 0.63 ?m and finally activated in methanol. The deionised water and Septonex (5•10-3 mol•l-1) at pH 8 were used for Amberlite conditioning. The optimal sorption of uranium (VI) proceeded at pH 8 in the presence of 4-(2-pyridylazo)resorcinol or ammonium pyrrolidinedithiocarbamate with Amberlite XAD-16 moreover with 1,2-dihydroxyanthraquinone-3-sulphonic acid. All organic reagents have mass concentration five time larger than uranium . For the elution of uranium the mixture of 1 mol•l-1 HNO3 with acetone (ratio of 1:1) was used. Acetone was evaporated and the final analysis performed using ICP-OES. The preconcentration of uranium (VI) using Silicagel-C18 conditioning was performed with ethanol, deionised water and Zephyraminu (5•10-4 mol•l-1) at pH 8. For the Silica-C8 and Silicagel-Phenyl the same conditioning procedure without zephyramin was used. Silica-C18 had the highest sorption efficiency with 1,2-dihydroxyanthraquinone-3-sulphonic acid, Silicagel-C8 with ammonium pyrrolidinedithiocarbamate and Silicagel-Phenyl with 8-hydroxyquinoline-5-sulfonic acid, all organic reagents have mass concentration five time larger than uranium concentration. As an optimal elution mixture acetone and ethanol (ratio of 1:1) in the presence of 1 mol•l-1 HCl was evaluated. Acetone and ethanol was evaporated and the final analysis was performed using ICP-MS. Silicagel-C18 was evaluate as the most effective in the presence of zephyramin and 1,2-dihydroxyanthraquinone-3-sulphonic acid. Sorption was also tested in the presence of 20 ?g•l-1 microelements (Be, V, Co, Ni, Y, Pb, Th, Cd) and in the presence of a defined concentrations of K, Na, Ca, Mg, Al and Fe. Direct analysis of uranium was carried out on samples of water from the river Ploucnice and Turonian aquifers containing uranium, 3,5 and 19,3 ?g•l-1. Different type of water required preconcentration of uranium (VI) on Silica-C18 in the presence of 1,2-dihydroxyanthraquinone-3-sulphonic acid and zephyramin. The analysis results gave statistically satisfactory results, which were confirmed using standard addition of 20 ?g•l-1.
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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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PRODUCTION OF BETA-GLUCANS AND OTHER POLYSACCHARIDES BY YEAST AND MICROALGAE
Byrtusová, Dana ; Kráčmar, Stanislav (referee) ; Kovalčík, Adriána (referee) ; Márová, Ivana (advisor)
Beta-glukany jsou polysacharidy složeny z monomerů D-glukózy. V dnešní době se -glukany těší zvýšené pozornosti zejména kvůli imunomodulační aktivitě a využitelnosti ve farmaceutickém a potravinařském průmyslu. Saccharomyces cerevisiae je dodnes jediným kvasinkovým zdrojem požívaným v biotechnologické produkci. Avšak některé kvasinky z oddělení Basidiomycetes, které jsou schopny produkce lipidů a karotenoidů, mohou být využity rovněž jako alternativní zdroj -glukanů. Dizertační práce se zabývá možností a optimalizací produkce -glukanů a dalších mikrobiálních sacharidů u karotenogenních kvasinek a mikrořas. Testovány byli zástupci rodů Rhodotorula, Sporobolomyces, Cystofilobasidium a Dioshegia. Z nekarotenogenních kvasinek byly do screeningu zařazeny kvasinky rodu Metschnikowia, askomycetní kvasinky a z mikrořas zástupci zelených a červených řas. Experimentální část cílí rovněž na možnosti koprodukce dalších metabolitů, jako jsou lipidy, pigmenty a extracelulární polymery. První část experimentu se zabývá vlivem čtyř C/N poměrů (10:1, 40:1, 70:1 a 100:1) na produkci biomasy, -glukanů, karotenoidů a lipidů. Ze všech testovaných kmenů, S. cerevisiae CCY 21-4-102, C. infirmominiatum CCY 17-18-4, P. rhodozyma CCY 77-1-1 a R. kratochvilovae CCY 20-2-26 vykazovaly nejvyšší produkci -glukanů a byly proto vybrány k podrobnější optimalizaci, zejména osmotického stresu, teploty a zdroje dusíku v kultivačním médiu. Dodatečně, kmen R. kratochvilovae CCY 20-2-26 je schopný produkce extracelulárních glykolipidů a S. pararoseus CCY 19-9-6 extracelulárních polysacharidů. Následně bylo stanoveno množství -glukanů u dalších dvanácti kmenů S. cerevisiae a rovněž možnost produkce polysacharidů u mikrořas.
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Complex characterisation of medical herbs and study of their utilization in food industry as a source of functional components
Burdějová, Lenka ; Kráčmar, Stanislav (referee) ; Šilhár, Stanislav (referee) ; Polovka, Martin (advisor)
Presented thesis deals with the complex characterization of extracts from medical plants using spectroscopic and chromatographic techniques, with emphasis on the influence of solvent, post-harvest treatment of the sample, location and year of production on selected parameters. Part of the work is devoted to application of selected extracts from medical plants in suitable form into beverages. The thesis is divided into two parts. At the first part of the work combination of electron paramagnetic resonance, ultraviolet visible spectroscopy, liquid chromatography and inductively coupled plasma optical emission spectrometry was applied for characterization of aqueous, ethanol and dimethylsulfoxide extracts from 10 selected medical plants, which were collected from two different locations during two years (2015, 2016) and post-harvestly treated by two ways (freezing, drying). In total, 39 parameters were determinated: namely total phenolic compounds content, total flavonoids content, 14 specific phenolic compounds, colour characteristics in the CIE L*a*b* system (L *, a*, b *, C*, h°, BI), radical-quenching activity using the cation radical of the 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid), antioxidant/pro-oxidant activity using the spin trapping technique in the presence of 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO)/potassium peroxodisulphate and content of 15 minerals (Al, As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, Zn). The results confirmed that the studied parameters of medical plants are influenced by the extraction system, post-harvest treatment of samples, year of production, origin of samples (locality) and species composition. It has been shown that the type of extraction solvent significantly affects the composition of the extracts. On the basis of these analyzes, for food application – the isolation of the main functional components (phenolic compounds, flavonoids) –50% ethanol, post harvest treatment by drying and samples of Mentha piperita, Melissa officinalis, Hypericum perforatum and Salvia officinalis were selected. The second part of the thesis was focused on the application of selected extracts of medical plants to beverages (syrups). The above mentioned plants, 25% ethanol instead of 50% ethanol (due to the limitation of the ethanol content in final raw material), were selected for the application. At first, extraction of medical plants was optimized. Suitable ratio of material and solvent (1:10), extraction time (8 h) and herb absorbency (50–62 %) were determined. The prepared extract after filtration was applied to the syrup concentrate in different ratios to produce one-species syrups and further sensorially tested to select the suitable recipe, the best flavour and basis for the production of two-species syrups. The mint syrup was the most tasty and generally the most acceptable one-species syrup, and sage syrup was selected as the basis for two-species syrups. Further, the most appropriate recipes of the two-species syrups were chosen, the most sensorially acceptable one was selected and further tested if it would be appropriate to colour them. Preferentially, it has been found that it is not necessary to colour two-species syrups. On the basis of the sensory analysis, it has been found that peppermint and sage-peppermint syrups were the most tasty and acceptable syrups, which could be, after further testing, produced industrially to enrich the food market.
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The Use of Separation Methods for the Study of Selected Drugs and Pesticides in Water
Burešová, Jitka ; Hajšlová, Jana (referee) ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
This work is focused on the study of penetration of pesticides and pharmaceuticals in the aquatic environment. The aim was to develop and verify the optimal method for long-term monitoring of the occurrence of selected pesticides and pharmaceuticals in surface waters. Two frequently used analgesics ibuprofen and diclofenac, and carbamazepine that is used to treat epilepsy, were chosen from the group of drugs. A group of 41 pesticides were selected for monitoring too. The attention was focused on a group of urea pesticides and phenoxyacetic acids, as well as on selected pesticides falling to the group of priority substances or identified as environmental quality standards. Some of pesticides were selected because their consumption in the Czech Republic exceeds 30 tons a year. Liquid chromatography with tandem mass spectrometry (LC / MS / MS) was used for the determination of selected analytes in surface waters. Two optimized analytical method were developed. Ten analytes were determined in negative ion mode and 34 analytes in positive ion mode. Solid phase extraction (SPE) was used for purification and concentration of the samples. Developed methodology was validated and used for monitoring of selected compounds in samples of surface waters from the Morava River basin. Samples were collected on the rivers Morava and Svratka monthly for two years. Samples from the river Blata and stream Nivnička were collected for one year. Surface water samples were also collected from the river Litava. Four samples were taken throughout the length of the flow during one day. Smaller streams were chosen for monitoring because of the probability of occurrence of pesticides. They flow abundantly agriculturally cultivated areas. Drugs ibuprofen and diclofenac were detected in all taken samples, carbamazepine mostly. Among the most commonly occurring pesticides were bentazon, diuron, isoproturon, carbendazim, MCPA, MCPP, propiconazole, tebuconazole and 2,4-D.
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The factors influencing sensory quality of processed cheese analogues
Sůkalová, Kateřina ; Kráčmar, Stanislav (referee) ; Diviš, Pavel (referee) ; Buňka,, František (advisor)
The presented thesis deals with the monitoring sensory quality of processed cheese analogues, focusing mainly on the taste (flavour) and related content of volatile (aroma active) substances. Model samples of analogues were produced by a standard procedure at Tomas Bata University in Zlín. The experimental part was divided into two experiments, which differed in the composition of model samples of analogues. In the first experiment, the traditional fat (butter) was completely replaced by selected vegetable fats (palm, coconut, mixed), in the second experiment only a part (1% w/w - expressed on the total weight of the sample) of butter was replaced by vegetable oils (apricot, flax seed, currant, grape seed). Solid phase microextraction in conjunction with gas chromatography with flame ionization detection was used to determine volatiles. Methods based on valid international standards were used to evaluate the sensory quality of samples, focusing mainly on flavor, namely evaluation using scales (ISO 4121), profile test (EN ISO 13299) and ranking test (ISO 8587). The aim of the work was to assess the effect of the addition of various vegetable fats/oils on the above parameters, at the same time their changes were monitored during 6 months of storage (at 6 ° C). The results showed that the vegetable oil used affects both the sensory quality and the content and composition of volatile substances of analogues. Significant differences between samples were mainly in taste, aroma and overall acceptability. The decreasing overall acceptability of the samples can be expressed in experiment I by the order: product with butter coconut palm mixed fat; in the case of experiment II: product with butter apricot = flax = grape currant oil. Mixed fat, currant and grape seed oil proved to be unsuitable for the production of analogues, on the contrary, analogues with coconut fat and apricot oil were evaluated best. Based on the results of sensory analyses, it was shown that the samples maintain a good sensory quality min. for 3 months while maintaining a low storage temperature ( 6 ° C). An analogue with coconut fat, whose taste, aroma and acceptability were rated as very good, and apricot oil, whose taste, aroma and acceptability were even rated as excellent, could enrich the food offer on the market.
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