|
|
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.
|
|
|
Use of stress factors for the production of lipid substances by yeasts of the genus Metschnikowia
Tručková, Marie ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
This Master´s thesis consists of two parts. The theoretical part is focused on the influence of various stress factors in the cultivation process, such as osmotic stress, oxidative stress, dehydration, cultivation time, pH or temperature on yeasts of the Metschnikowia genus. The practical part deals with osmotic and oxidative stress. Oxidative stress was performed under two different conditions (various cultivation time and temperature). The properties of yeast production were monitored mainly by gas chromatography and marginally also by flow cytometry. The analysis was performed for Metschnikowia pulcherrima, Metschnikowia andauensis, Metschnikowia chrysoperlae, Metschnikowia sinensis, Metschnikowia zizyphicola and Metschnikowia shanxiensis strains. The results showed that both oxidative stress and osmotic stress induce better lipids production. The amount of lipids, especially the fatty acid composition, also varied depending on the strains studied and the culture conditions used. The production of unsaturated fatty acids was further demonstrated in this work. The most suitable medium for the production of lipids and unsaturated fatty acids was the salt medium. It is clear that yeasts of the genus Metschnikowia are highly adaptable yeasts. Therefore, they might be potentially auspicious biotechnology producers.
|
|
|
Metabolic and biophysical characterization of bacterial cells capable of PHA accumulation
Slaninová, Eva ; Lehocký, Marián (referee) ; Doškař, Jiří (referee) ; Chodak, Ivan (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.
|
|
|
Tolerance Pavlownia tomentosa in vitro k suchu a osmotické zátěži
Zapalač, Radek
This diploma thesis deals with the influence of abiotic stressors on plant growth and their acclimation responses. An in vitro culture experiment was conducted with Paulownia tomentosa plants for 35 days, in which growth parameters (reduction of leaf turgescence, leaf damage, plant height, number of nodes per stem, number of leaves and number of roots) and plant survival, under the treatment of three different osmotics at different concentrations in the medium (20 g/l sucrose – control, 80 g/l sucrose, 50 mM NaCl, 100 mM NaCl, 1.5 % PEG6000, 3 % PEG6000). On days 10 and 30 of the experiment, plant samples were collected for determination of ABA and proline content in leaves. The radioimmunoassay method was used to determine ABA content, while proline content was determined by spectrophotometric measurement. During the first ten days of cultivation, gas samples were also taken from the culture vessels to determine ethylene production, which was subsequently carried out by gas chromatography. The results obtained were statistically and graphically processed and supplemented by photodocumentation.
|
|
|
Mitochondria morphology and membrane potential under stress conditions
Ezati, Masoumeh ; Zumberg, Inna ; Cmiel, Vratislav
Mitochondria, organelles found in the cytoplasm of almost all eukaryotic cells, generate large quantities of energy in the form of adenosine triphosphate (ATP). In addition to producing energy, mitochondria store calcium for cell signaling activities and play an important role in maintenance of ionic balance. Mitochondria, however, are highly sensitive to any kind of stress in which they mainly response by disturbance of respiration, reactive oxygen species (ROS) production and release of cytochrome c into the cytoplasm. Osmotic stress, in particular, generates ROS that degrade lipids, proteins, and DNA. High levels of salt concentration can cause an imbalance in cellular ion homeostasis that results in ion toxicity and osmotic stress. This study aims to investigate possible effects of KCl and NaCl on Bone marrow-derived (MSCs) mitochondria membrane potential (MMP) and morphology. The results indicated that KCl and NaCl of salt concentration can cause an imbalance in cellular ion homeostasis that results in ion toxicity and osmotic stress. This study aims to investigate possible effects of KCl and NaCl on MSCs mitochondria membrane potential (MMP) and morphology. The results indicated that KCl and NaCl decreased the potential and changed the morphology of mitochondria membrane compared to cells growing in normal condition.
|
|
|
Experimental cultivation systems in root system research
Střelec, Petr ; Soukup, Aleš (advisor) ; Lhotáková, Zuzana (referee)
The growing awareness of plant root importance has contributed to the continuous development of cultivation methods that facilitate more efficient and effective root research. Due to their sessile nature, plants rely extensively on roots for water and ion absorption (Meister et al., 2014). As a result, plant roots must adjust to an array of unfavorable conditions such as salinity, drought, and high temperatures. To better understand root response and promote agricultural development, researchers simulate various stressors in controlled conditions. More recently developed transparent soils and soilless cultures aim to overcome the limitations of traditional soil cultivation, which include imprecise variable control, heterogeneous conditions, and destructive root sampling (Gregory et al., 2009). When planning an experiment, it is crucial to select an appropriate cultivation method to ensure success. The aim of this thesis is to provide a comprehensive overview of the benefits and limitations of commonly used cultivations techniques in plant stress research. Transparent soil reliably mimics the mechanical properties of soil whilst allowing for easy root access and use of numerous visualization techniques. On the other hand, it still maintains the need for a larger cultivation area just like soil. The...
|
|
|
Use of stress factors for the production of lipid substances by yeasts of the genus Metschnikowia
Tručková, Marie ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
This Master´s thesis consists of two parts. The theoretical part is focused on the influence of various stress factors in the cultivation process, such as osmotic stress, oxidative stress, dehydration, cultivation time, pH or temperature on yeasts of the Metschnikowia genus. The practical part deals with osmotic and oxidative stress. Oxidative stress was performed under two different conditions (various cultivation time and temperature). The properties of yeast production were monitored mainly by gas chromatography and marginally also by flow cytometry. The analysis was performed for Metschnikowia pulcherrima, Metschnikowia andauensis, Metschnikowia chrysoperlae, Metschnikowia sinensis, Metschnikowia zizyphicola and Metschnikowia shanxiensis strains. The results showed that both oxidative stress and osmotic stress induce better lipids production. The amount of lipids, especially the fatty acid composition, also varied depending on the strains studied and the culture conditions used. The production of unsaturated fatty acids was further demonstrated in this work. The most suitable medium for the production of lipids and unsaturated fatty acids was the salt medium. It is clear that yeasts of the genus Metschnikowia are highly adaptable yeasts. Therefore, they might be potentially auspicious biotechnology producers.
|
|
|
Water use efficiency (WUE), its definition and importance in plant resistance against osmotic stress
Jedlička, Tomáš ; Hála, Michal (advisor) ; Lhotáková, Zuzana (referee)
Water is one of the main limiting factors of plant growth. Availability of water on our planet is decreasing, and if we add fast-growing human population, we have a problem growing. Scientists from all around the world, especially those from arid regions, are facing a big problem, which is an attempt to make the most efficient use of water by plants. If this problem should be solved, we need to understand the issue of Water Use Efficiency (WUE). This paper attempts to summarize the current knowledge related to the issue of WUE, mainly focused on anatomical and physiological parameters control of WUE, which is focused primarily on the issue of evapotranspiration. Another part focuses on clarifying the issue of WUE at the gene level and the related biotech opportunities. At the end of the work are discussed possibilities of increasing WUE using irrigation techniques. Key words: water use efficiency, evaporation, transpiration, stoma, stomatal density, abscisic acid, ERECTA
|
|
|
Role of the yxkO gene of Bacillus subtilis in responce to environmental stress.
Petrovová, Miroslava ; Lichá, Irena (advisor) ; Nešvera, Jan (referee)
ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.
|
|
|
Metabolic and biophysical characterization of bacterial cells capable of PHA accumulation
Slaninová, Eva ; Lehocký, Marián (referee) ; Doškař, Jiří (referee) ; Chodak, Ivan (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.
|
guest ::
