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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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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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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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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Stress resistance of PHA accumulating bacteria against conditions associated with gastrointestinal tract
Kovářová, Radka ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.
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Study on PHA production by selected thermophillic bacteria
Brondová, Zuzana ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of thermophilic bacterial strains Tepidiphilus thermophilus and Chelatococcus daeguensis aimed at the production of polyhydroxyalkanoates (PHA). Experiments were developed to determine properties of use of these microorganisms in the biotechnological industry. In the experimental parts, the presence of the phaC gene indicating PHA production was confirmed by both bacteriological strains by polymerase chain reaction (PCR). Subsequently, the conditions for PHA production were optimized. Condition profiling aimed at determining the optimal temperature, optimal carbon source, and the effect of the precursor on cell growth, composition and amount of PHA through GC-FID. The last experiment was to determine the utilization of selected substrates. The optimal conditions for the Tepidiphilus thermophilus strain were temperature above 55 ° C and glycerol as the best carbon source. Precursors inhibited cell growth, but the highest amount of 3HV was determined when valeric acid was added to medium. The Chelatococcus daeguensis strain had an optimal temperature of 45 ° C and glucose was optimal carbon source. The best cell metabolizing precursor was 1,4-butanediol. Both bacterial strains were capable of utilizing many substrates where glucose and valeric acid were best.
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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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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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Utilization of PHA producing bacteria in bioremediation technologies
Šuráňová, Zuzana ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work is study of utilization of PHA producing bacteria in bioremediation technologies. For this study were used bacteria Pseudomonas putida KT2440 and two isolates from soil contaminated by petroleum - Pseudomonas gessardii (D2) a Pseudomonas fulva (D3). The experimental part describes especially study of feather biodegradation using selected microbial strains. All the tested bacterial strains were capable of feather degradation and utilization as the sole carbon source. During biodegradation experiment, we monitored weight loss of feather, protease and keratinase activity, concentration of bacterial biomass and PHA content as well as pH. The highest biodegradation ability and keratinase activity was observed in Pseudomonas putida. None of tested bacteria accumulated detectable amount of PHA during growth on waste feather, nevertheless, bacterial biomass grown during feather degradation can be used as an inoculum for PHA production on waste frying oil and octanoic acid. Using this experimental setup, high PHA content (54% of cell dry weight) was achiaved in Pseudomonas putida. Another part of the thesis deals with biodegradation of petroleum oil. The highest capability of growth on this carbon source were determined in Pseudomonas fulva.
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Connection between stress resistance of bacteria to temperature-mediated stress and PHA accumulating ability
Nováčková, Ivana ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the involvement of polyhydroxyalkanoates (PHA) into the stress response of bacteria, specifically when they are exposed to temperature-mediated stress. The theoretical part focuses on the stress response of bacteria in general, the mechanism of response to temperature-mediated stress and PHA involvement into the stress response. The aim of experimental work was to study the correlation between the amount of PHA in the biomass of the bacteria and their viability after exposure to temperature-mediated stress. PHA producing bacterial strains Cupriavidus necator H16, Burkholderia cepacia and Burkholderia sacchari and non-producing mutant strain Cupriavidus necator PHB4 were used for experimental work. Bacteria accumulated poly(3-hydroxybutyrate) (PHB), the amount of PHB in the biomass was determined by gas chromatography with FID. Viability of live bacterial cells and cells after exposure to temperature-mediated stress was determined by using flow cytometry. It turned out that the content of PHB in biomass assists bacteria to resist the freezing shock. By comparing the viability of bacteria C. necator H16 and C. necator PHB4 after exposure to multiple stresses it also has been observed that PHB increased the resistance of cells against temperature-mediated stress and stress caused by low pH.
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