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Biocrystallization as a new concept in development of piezoelectronics for use in biomaterials
Dobešová, Kateřina ; Plichta, Tomáš (referee) ; Sedláček, Petr (advisor)
This thesis focuses on biofilament methods and biocrystallization of bacterial cells producing polyhydroxyalkanoates, specifically the bacterium Cupriavidus necator H16. The main techniques used for cell orientation include spin-coating and acoustic waves. Biocrystallization was induced by temperature, osmotic, and acid stress. The degree of cell orientation was monitored by an atomic force microscope, and crystallization was determined by FTIR. Among other parameters, the thickness of the layers was evaluated using a profilometer. The amount of PHA in the bacterial cells was determined by gas chromatography. The preparation of crystalline ordered layers of biomaterials represents a route to piezoelectric biomaterials.
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The influence of electrochemically generated hydrogen peroxide on the stress response of microorganisms for the purpose of increased PHA production
Najbrtová, Johana ; Nováčková, Ivana (referee) ; Ehlich, Jiří (advisor)
The subject of the presented thesis is the effect of electrochemically formed hydrogen peroxide on the production of polyhydroxyalkanoate. In this work, hydrogen peroxide was generated directly in the medium with bacteria by two-electron reduction of oxygen. This thesis is divided into a theoretical part, which is devoted to approaching the topic of polyhydroxyalkanoate, oxidative stress, electrochemical production of H2O2 and the connection of electrochemistry with biotechnology. The experimental part is focused on the application of these findings. Hydrogen peroxide was generated by applying an electrical potential to an electrode system composed of a stainless-steel working electrode, a titanium counter electrode, and a pseudo-reference Ag/AgCl electrode embedded in a polypropylene holder. After initial electrochemical characterization of the prepared system, a potential of 0,8 V and -1 V was selected for application in cultivation experiments. Here, the effect of H2O2 on the stress response of the bacterium Cupriavidus necator H16 from the Czech collection of microorganisms was investigated. From the results of the cultivation experiments, it was evident that the electrochemically produced H2O2 does not have a significant effect on the development of the bacterial culture. However, the electrode system itself positively affects bacterial growth. The influence of H2O2 was not noticeable due to the low production in medium. This was caused by a low concentration of oxygen in the medium. During this work, a new electrode system with higher H2O2 production was designed, which could use atmospheric oxygen for reduction to H2O2. However, even with this system, a desired stress response was not achieved, because increased production of H2O2 was prevented by the formation of biofilm on the electrodes. Furthermore, it was found that the materials used for the electrode system support the formation of biofilm, mainly titanium, stainless-steel mesh and polypropylene.
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Characterization of bacterial strains obtained in evolutionary engineering
Hrabalová, Vendula ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with application of evolutionary engineering on PHA producing bacterial strains. Two bacterial strains, Cupriavidus necator H16 and Halomonas halophila, were chosen for the evolutionary experiments. Copper cations (Cu2+) and sodium chloride (NaCl) were chosen as the selective pressure for C. necator H16; acetic acid (AA) and levulinic acid (LA) for Halomonas halophila. The adapted strains were during long-time evolutionary experiments characterized by GC-FID and SEC-MALS. The growth of the adapted strains was studied by the mean of optical density measurement. The amount of viable cells was determined by spectral FC after their expositon to selected stress factors. Specific enzyme activities of enzymes involved in citrate and glyoxalate cycle, enzymes generating NADPH, LA metabolism enzyme and PHA biosynthesis enzymes were determined. The adapted strains were compared with the wild-type of strains. The successfull adaptation of C. necator H16 adapted to Cu2+ was detected. Biomass and PHA production of both wild and adapted H. halophila strains cultivated in lignocellulosis waste were determined. It was found out that H. halophila adapted to the LA is capable of producing more PHA than the wild strain of this bacteria.
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Acetic acid as a side product of metabolism of PHA producing bacteria
Ryšavá, Lenka ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
The Bachelor´s thesis deals with experimental study production of acetic acid as a side product of metabolism of PHA producing bacteria. Experimental part of work deals with the impact of used substrate on the biomass growth, PHA production and the formation of organic acids in strains that can potentially find their application in industrial production of PHA – Cupriavidus necator H16, Burkholderia sacchari, Burkholderia cepacia. The highest yields of organic acid have been reached on Burkholderia sacchari when cultivated on mineral medium with glucose. The last part of the thesis is focused on determining the viability of micro organisms using flow cytometry where bacteria Cupriavidus necator H16, Burkholderia sacchari, Burkholderia cepacia were exposed to selected stress factors.
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Involvement of polyhydroxyalkanoates in stress response of bacteria during late stationary phase
Šuráňová, Zuzana ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase. Bacteria Cupriavidus necator H16 (able to produce PHA) and bacteria Cupriavidus necator H16/PHB-4 (unable to produce PHA) were used for the experiment. In the theoretical part, the polyhydroxyalkanoates and a stress response of bacteria were reviewed. In the experimental part of the work, the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase against selected stress factors was studied. A resistence against various stress conditions of bacteria was studied. During long term cultivations a culture viability as well as PHA distribution among bacterial populations were assessed by using flow cytometry and the PHA content in biomass was analyzed by gas chromatography with FID detector.. Based on the results obtained in this work, it was found that the PHA acumulating bacteria Cupriavidus necator H16 is capable to survive carbon substrate limitations better than the bacteria Cupriavidus necator H16/PHB. Further, Cupriavidus necator H16 also revealed higher resistence against various stress factors such as ethanol treatment and freezing.
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Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
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Production of PHB-HV from grape pomace by employing a pure culture of Cupriavidus necator
Jakešová, Michaela ; Kučera, Dan (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deal with the production of PHB-HV copolymer employing a pure culture of Cupriavidus necator H16. First, Erlenmeyer flask´s cultivations using pure fructose as a source of carbon were performed. The mode of addition of valeric acid for the synthesis of PHB-HV was studied. Furthermore, the effect of stress factor (ethanol) on PHB-HV synthesis by this microorganism was investigated. The method obtained by optimization in Erlenmeyer flasks was applied to the synthesis of the PHB-HV copolymer in the bioreactor using fructose as a source of carbon, too. Furthermore, after enzymatic hydrolysis, a sugar extract from grape pomace was obtained as a source of carbon for the synthesis of PHB-HV. The fermentation in Erlenmeyer flasks and the bioreactor using an extract of grape pomace was carried out. Using this alternative carbon source 3.51 g/l PHB-HV copolymer was produced in Erlenmeyer flask, 5.06 g/l in bioreactor respectively. In the first case, the proportion of 3-hydroxyvalerate in the copolymer was 23.78 mol. % and in the second one, 9.39 mol. %.
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Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
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Involvement of polyhydroxyalkanoates into stress response of bacteria
Kučera, Dan ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates (PHA) into stress response of bacteria. The theoretical part of the thesis deals with the possibility of establishing the viability of microorganisms using modern techniques, in particular flow cytometry. Furthermore, the research focused on selected stress factors and PHA involvement in stress response was described. In the experimental part of the work the stress response with regard to the ability to accumulate PHA was assessed. Bacteria Cupravidus necator was used for the experiment. Its ability to accumulate PHA at a later stage of growth increased resistance to ethanol, high temperature and freezing. Conversely, the PHA-producing strain showed lower resistance to the action of inorganic acids and bases. This may be caused by different morphology of PHA-producing cells. One of partial objectives was also to study the possibilities of staining of living cells accumulating PHA using Nile red. The research proved that the dye penetrates into living cells at elevated temperature of 40-45°C. This temperature is not lethal to the cells and the intensity of staining is sufficient to distinguish PHA-producing cells using flow cytometry; that can be applied in the selection of industrial PHA producers.
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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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