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Biotechnological production of PHA by selected bacterial isolates
Matějka, Filip ; Šedrlová, Zuzana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the production of polyhydroxyalkanoates (PHA) using thermophilic bacterial isolates with designation 34, 35 and BŽ. Bacterial straines were isolated from activated sludge and compost The theoretical part contains a description of PHA, extremophilic bacteria and detection techniques for the determination of polyhydroxyalkanoates. In the experimental part, the presence of the phaC gene, which is crucial for the ability to produce PHA, was first determined by PCR and bacterial strains were also subjected to DNA sequencing of 16S rRNA gene which enabled preliminary taxonomical classification of the isolates. In the next part of the experimental work, the conditions for biomass growth and PHA production were optimized. Suitable carbon source, the ideal temperature for cultivation and the influence of precursors on the production of copolymers were studied and identified. The composition and proportion of PHA were determined spectrophotometrically and by GC-FID. Finally, visual screening of PHA accumulation inside bacterial cells was performed using fluorescence microscopy.
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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
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production of PHA by selected extremophilic bacteria
Šarköziová, Patrícia ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor´s thesis deals with production of polyhydroxyalkanoates (PHA) by thermophilic bacterial strains of Chelatococcus composti and Chelatococcus sambhunathii. We were finding out the best conditions for production of PHA granules inside the cells. In the beginning of the experiment we employed PCR to detect the phaC gene, encoding for PHA synthase, which is requisite for production of PHA. We also analyse the best source of carbon, optimal temperature for production of polyhydroxyalkanoates and influence of chosen precursors to grow of biomass and production of copolymers. The last part of this experimental thesis deals with research utilization of selected model hydrolysates. Glucose was the best source of carbon for both bacteria Chelatococcus composti and Chelatococcus sambhunathii and optimal temperature for grow and production of P(3HB) was 45°C. The applied precursors inhibited cell grow, but both bacterial strains Chelatococcus composti and Chelatococcus sambhunathii could produce copolymer P(3HB-co-3HV) after additiong of precursors sodium propionate and valeric acid. For both bacterial strains was the most useful model hydrolysates, which contains the biggest part of glucose. Morever, bacterial cells containing PHA granules were observed by fluorescence and transmission electron microscopy.
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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
|
|
|
Biotechnological production of PHA by selected bacterial isolates
Matějka, Filip ; Šedrlová, Zuzana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the production of polyhydroxyalkanoates (PHA) using thermophilic bacterial isolates with designation 34, 35 and BŽ. Bacterial straines were isolated from activated sludge and compost The theoretical part contains a description of PHA, extremophilic bacteria and detection techniques for the determination of polyhydroxyalkanoates. In the experimental part, the presence of the phaC gene, which is crucial for the ability to produce PHA, was first determined by PCR and bacterial strains were also subjected to DNA sequencing of 16S rRNA gene which enabled preliminary taxonomical classification of the isolates. In the next part of the experimental work, the conditions for biomass growth and PHA production were optimized. Suitable carbon source, the ideal temperature for cultivation and the influence of precursors on the production of copolymers were studied and identified. The composition and proportion of PHA were determined spectrophotometrically and by GC-FID. Finally, visual screening of PHA accumulation inside bacterial cells was performed using fluorescence microscopy.
|
|
|
production of PHA by selected extremophilic bacteria
Šarköziová, Patrícia ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor´s thesis deals with production of polyhydroxyalkanoates (PHA) by thermophilic bacterial strains of Chelatococcus composti and Chelatococcus sambhunathii. We were finding out the best conditions for production of PHA granules inside the cells. In the beginning of the experiment we employed PCR to detect the phaC gene, encoding for PHA synthase, which is requisite for production of PHA. We also analyse the best source of carbon, optimal temperature for production of polyhydroxyalkanoates and influence of chosen precursors to grow of biomass and production of copolymers. The last part of this experimental thesis deals with research utilization of selected model hydrolysates. Glucose was the best source of carbon for both bacteria Chelatococcus composti and Chelatococcus sambhunathii and optimal temperature for grow and production of P(3HB) was 45°C. The applied precursors inhibited cell grow, but both bacterial strains Chelatococcus composti and Chelatococcus sambhunathii could produce copolymer P(3HB-co-3HV) after additiong of precursors sodium propionate and valeric acid. For both bacterial strains was the most useful model hydrolysates, which contains the biggest part of glucose. Morever, bacterial cells containing PHA granules were observed by fluorescence and transmission electron microscopy.
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