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Utilization of selected extremophilic bacteria for bioplastic production
Vidláková, Michaela ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on the study of the biotechnological potential of thermophilic bacteria Tepidiphilus thermophilus, Tepidimonas taiwanensis and Tepidimonas fonticaldi. The aim of this work was to investigate the ability of bacteria to produce polyhydroxyalkanoates (PHA). The theoretical part includes the characteristics of PHA and extremophilic bacteria. In the experimental part, the investigated bacteria were tested by molecular technique PCR for the presence of the phaC gene encoding for the first class PHA synthase. The presence of the phaC gene was confirmed in bacterial strains Tepidiphilus thermophilus and Tepidimonas taiwanensis. The ability of PHA production on various carbon sources was examined for all the selected microorganisms, optimal temperatures for cultivation were determined, and the ability to synthesize copolymers was investigated by adding suitable precursors to the production medium. The most intensive PHA production was recorded in the bacterial strain Tepidimonas taiwanensis, but other strains were also able to produce PHA. The ability to produce P(3HB-co-3HV) or P(3HB-co-4HB) copolymers has also been demonstrated in some strains. A significant amount of P(3HB-co-3HV) was obtained using the Tepidimonas taiwanensis strain. Morever, Tepidimonas taiwanensis strain was also tested for its ability to use various model lignocellulose hydrolysates to form PHA. The highest PHA gain was obtained using model hydrolysate of softwood. Furthermore, the ability of proteases production by selected bacterial strains was tested. The presence of these enzymes was detected in the Tepidimonas taiwanensis strain, which was followed by determination of the temperature optima of the proteases, the pH optima of the proteases and also of their molecular weights.
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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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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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Isolation, identification and characterization of extremophiles capable of PHA production
Vlasáková, Terézia ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.
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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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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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