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Adaptation to adverse osmotic conditions as a tool for evolutionary enginnering of bacteria
Drotárová, Lenka ; Nováčková, Ivana (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with the application of osmotic stress as a tool for evolutionary engineering of PHA producing bacterial strains. The aim of this thesis is to evaluate a bacterial adaptation to hypoosmotic environment, as an engineering tool in order to increase the production of PHA. The theoretical part focuses on the evolutionary engineering principle, methods of the strategy and the effect of physical factors on microorganism. The aim of experimental part was to performed an adaptive evolutionary experiment with the bacterial strain Halomonas halophila CCM 3662. Reduced osmotic pressure was used as a stressing factor during the serial cultivation. In order to generate PHA producing mutant strains, each passage was characterized using spectrophotometric and gravimetric method and by GC-FID. It was found that after the long-term cultivation, the mutant strain HH35, cultivated in 35 g/l NaCl, was associated with the highest biomass and PHB concentration. The 15th and 30th passages, along with the wild type strain H. halophila were subjected to further cell-robustness analysis with the application of hyper- and hypoosmotic shock. The stress response, viability of cells and morphological changes were analyzed using FC and TGA methods. Isolated polymers were characterized using FTIR analysis.
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Utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of PHA producing bacteria
Beránková, Barbora ; Enev, Vojtěch (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the study of the utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of polyhydroxyalkanoates (PHA) producing bacteria. Using gas chromatography with FID detection, we determined the polyhydroxybutyrate (P(3HB)) content of the PHA biomass of bacterial strains Burkholderia cepacia, Halomonas halophila, Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 and Lactobacillus delbrueckii, which is not a producer of polyhydroxyalkanoates but this bactrea was selected as representative of Gram-positive bacteria. Subsequently, thanks to Raman microspectroscopy, Raman tweezers and FT-IR spectrometer in combination with Raman FT-module, we were able to confirm or disprove the presence of P(3HB) in bacteria. Furthermore, the thesis describes Cupriavidus necator H16, which is a model organism for the production of P(3HB), and his mutant strain Cupriavidus necator H16/PHB-4. The bacterial strain Cupriavidus necator H16 was cultivated in a production mineral medium of various nitrogen contents, while cultivation was also carried out in liquid Nutrient Broth. By this cultivation we were able to reach various P(3HB) content in bacterial biomass, the spectra were subsequently compared with the spectrum of the bacterial strain Cupriavidus necator H16/PHB-4. Raman spectroscopy is well used to characterize the composition of individual bacterial cells, is a fast, versatile, and virtually non-invasive tool for studying cells.
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Study of biological samples using atomic force microscopy
Khýrová, Markéta ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.
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Development of microbioreactor for microbial biotechnology
Jakešová, Michaela ; Szotkowski, Martin (referee) ; Krejčí,, Jan (advisor)
This master thesis deals with the development of a minibioreactor for microbial biotechnologies. The AlgaTox system – an analytical photobioreactor from BVT Technologies – was the default unit of the new device. The working volume of the new minibioreactor is in the range of 4 to 8 ml. The minibioreactor was composed of a minithermostat, a reaction vessel, an oxygen electrode, a temperature and pH probe, accessories for the supply of air to the liquid and an insertion for the transport of liquid from / to the reactor. The functionality of the assembled device and its characteristics were measured. Furthermore, an operating procedure for the decontamination process using a hydrogen peroxide mist was developed for the new equipment. An operating procedure for culturing microorganisms in a minibioreactor was also set up. Pilot cultivations of Halomonas halophila were demonstrated in the prepared equipment. In these cultivations, three assemblies for air supply to the liquid were tested. However, none of the assemblies was able to provide a sufficient supply of oxygen to cell culture – the dissolved oxygen value always dropped to 0%. For the further development, a new assembly was designed for the supply of air to the liquid - an aeration ring from a membrane tube.
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Isolation of PHA from bacterial biomass and their subsequent purification
Zlochová, Barbora ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the isolation and characterization of the bacterial polymer PHA isolated from the bacterial strain Halomonas halophila (CCM 3662) by osmotic stress in distilled water, ie hypo shock, when the cells crack and release PHA. The addition of biosurfactants simultaneously solubilizes the proteins and lipids of the residual biomass, theoretically achieving a purer PHB product without the use of toxic substances such as chloroform. The theoretical part of the work focuses on PHA in terms of their general properties and use, the mechanism of synthesis of PHA microorganisms and their subsequent degradation and the existing strategy for isolating PHA from bacterial biomass. The experimental part describes the production of PHB bacteria Halomonas halophila and the subsequent process of isolation of PHB by hypo shock of halophilic bacteria in distilled water with the addition of selected biosurfactants. Biosurfactants used in the work are coconut soap, lecithin, rhamnolipids, sophorolipids, coco glucoside, lauryl glucoside and cocoamidopropyl betaine. The properties of the isolated PHB material were analyzed by gas chromatography with FID detection and SEC-MALS and FTIR methods. Based on the results, we chose the pair of coco glucoside and lauryl glucoside as the most suitable biosurfactants that can be used to isolate PHB from bacterial biomass. In combination with washing the material in ethanol, at concentration of biosurfactant 5 g/l and isolation in 70 ° C, it was possible to achieve a purity of 90 % of material using cocot glucoside and 89 % purity of material using lauryl glucoside.
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Use of microcalorimetric techniques in the study of stress adaptation of microorganisms
Korfantová, Stanislava ; Slaninová, Eva (referee) ; Krouská, Jitka (advisor)
This bachelor thesis uses microcalorimetric techniques to study stress adaptation of wild strain of H. halophila and H. halophila strain adapted to lower salinity of medium in the environment of levulinic acid, which occurs naturally in hydrolysates of lignocellulosic materials. These can serve as inexpensive substrates on which H. halophila is capable of producing polyhydroxybutyrate. Microcalorimetric measurements showed that the wild-type H. halophila strain could adapt to levulinic acid to a concentration of 5 g/l. The adapted strain, which was assumed to have poorer stress management based on lower PHB yields, could adapt to all observed concentrations. The results were compared with a conventional method that confirmed the results of microcalorimetry. Microcalorimetry has been evaluated as a universal and susceptible method that is suitable for studying the metabolic activity of microorganisms. To better interpret the obtained microcalorimetric results, it is necessary to supplement the technique with information from other analytical techniques. The bachelor thesis uses spectrophotometric and gravimetric determination and gas chromatography with a flame ionization detector to characterize the wild and adapted strain before monitoring the adaptation to levulinic acid. The techniques offered enriching information to supplement the microcalorimetry outputs.
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Evolutionary engineering of PHA producing bacterium Halomonas halophila
Ikrényiová, Terézia ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with evolutionary engineering of PHA producing bacteria, the principle of PHA production and solution of this production’s disadvantages, but also applications of these biopolymers in the theoretical part. The production of polyhydroxyalkanoates by bacteria Halomonas halophila, which is focused on gaining the maximus amount of 3-hydroxyvalerate in formed copolymer, is described in the experimental part. The precursor valeric acid was added to bacteria due to gain the amount of 3HV. It was found that the concentrations over 3 g/l aren´t usable for production sufficient concentration of PHA. The very low concentrations of valeric acid led to low amount of 3HV in PHA. The available concentration of this precursor for production sufficient concentration of PHA by bacterial cell is 3 g/l. Moreover, it was found that the valeric acid should be added after 24 hours of cultivation in mineral production medium. The thesis is also concentred on comparison the original bacterial strains of Halomonas halophila to strains, which were adapted on valeric acid as stress factor for bacteria. The assumption, that the adapted strains can better utilize valeric acid and the incorporation to copolymer of it is higher like the original strains, was affirmed.
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Utilization of thermal analysis in the study on effects of microbial inhibitors
Bošeľová, Miriam ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the use of thermal analysis in the study on effects of microbial inhibitors. The main aim of this work was to determine the utilization of the method, which is mainly used in different fields of science and research. Three bacterial strains: Cupriavidus necator H16, its mutant strain Cupriavidus necator PHB-4 and Halomonas halophila, were used as model microorganisms. The inhibitory effect of levulinic acid on growth and metabolic activity was monitored by microcalorimetry. It was found that bacteria were able to adapt to levulinic acid to a certain concentration - Cupriavidus necator to 5 g/l and Halomonas halophila to 2 g/l. The thermal analysis results were compared to a conventional method, which is commonly used to study the growth of microorganisms.
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Biotechnological production of polyhydroxyalkanoates on wheat bran
Guziurová, Pavlína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotechnological production of polyhydroxyalkanoates (PHA) from lignocellulosic waste – wheat bran. The aim was to determine whether the hydrolysate from wheat bran subjected to different types of pretreatment could be used as a substrate for the production of PHA using selected bacterial strains. The selected strains were Halomonas organivorans, Halomonas halophila and two strains of Schlegelella thermodepolymerans. Halophilic strains were proved as the best producers and were subsequently used for cultivations. The hydrolysates after neutral pretreatment were utilized by the bacteria most efficiently, due to the lowest content of microbial inhibitors (phenolic substances) where the bacteria produced the most PHA. The highest value of produced PHA was determined on the hydrolysate after neutral pretreatment by using the strain Halomonas organivorans, namely 2,82 g/l. The hydrolysate was also used for the production of lactic acid bacterial strains of Lactobacillus. The highest achieved concentration of produced lactic acid was 16,73 g/l by the Lactobacillus casei strain.
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Utilisation of fluorescence techniques for analysis of industrially relevant microorganisms
Müllerová, Lucie ; Kráčmar, Stanislav (referee) ; Krčma, František (referee) ; Obruča, Stanislav (advisor)
Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodidem. Tento protokol byl také využit pro studium morfologie PHA-syntetizující halofilní bakterie Halomonas halophila. Pro stanovení UV-protektivních vlastností PHA obsaženého v bakterii Cupriavidus necator, byl optimalizován další protokol, tentokrát pro ROS citlivou sondu CM-H2DCFDA, která byla stanovená jako citlivější v porovnání s její nemethylovanou formou. V této studii byly navíc potvrzeny UV-protektivní vlastnosti PHA na bakterii Cupriavidus necator H16 v porovnání s PHA nesyntetizujícím kmenem Cupriavidus necator PHB-4.
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