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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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Biotransformation of ferulic acid to sensory active compounds
Opial, Tomáš ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotransformation of ferulic acid to sensory active substances. The experiment was based on monitoring the biotransformation potential of selected microorganisms by high performance liquid chromatography (HPLC) method while the cultivation was performed in two parallel runs and samples with added ferulic acid were divided into 24-hour effect and 48-hour effect of ferulic acid on bacterial cultures. Thermophilic bacteria and halophilic bacterium Halomonas neptunia have been found to be the most suitable candidates for the biotransformation of ferulic acid to sensory active substances. In both samples of Schlegelella thermodepolymerans (DSM 15344 and DSM 15264) was formed 33 mg/l and 76 mg/l of vanillic acid and 81 mg/l and 71 mg/l of 4-vinylguaiacol after 24 hours of ferulic acid effect. In a sample with T. taiwanensis was formed 61 mg/l of vanillic acid and 32 mg/l of 4-vinylguaiacol after 48 hours of ferulic acid effect, and in a sample of R. xylanophilus was formed 56 mg/l of 4-vinylguaiacol. In the sample with H. neptunia after 24 hours of ferulic acid effect was formed 296 mg/l of vanillic acid, which was up to 59% of the conversion of the added ferulic acid and in a sample with H. organivorans after 24 hours of ferulic acid effect was formed 71 mg/l of vanillic acid. However, after 48 hours of ferulic acid effect, vanillic acid degraded. In addition to screening of biotransformation potential, the sequence of bacterial enzymes, involved in biotransformation, was also compared with protein sequences in the database using the BLAST search tool. The presence of genes encoding enzymes involved in biotransformation has been demonstrated for almost all used microorganisms except H. neptunia, for which no gene has been identified. The highest number of genes present in bacteria was with the enzymes feruloyl-CoA synthetase, enoyl-CoA hydratase/isomerase, acetoacetyl-CoA thiolase and vanillin dehydrogenase.
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Biotransformation of ferulic acid to sensory active compounds
Opial, Tomáš ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotransformation of ferulic acid to sensory active substances. The experiment was based on monitoring the biotransformation potential of selected microorganisms by high performance liquid chromatography (HPLC) method while the cultivation was performed in two parallel runs and samples with added ferulic acid were divided into 24-hour effect and 48-hour effect of ferulic acid on bacterial cultures. Thermophilic bacteria and halophilic bacterium Halomonas neptunia have been found to be the most suitable candidates for the biotransformation of ferulic acid to sensory active substances. In both samples of Schlegelella thermodepolymerans (DSM 15344 and DSM 15264) was formed 33 mg/l and 76 mg/l of vanillic acid and 81 mg/l and 71 mg/l of 4-vinylguaiacol after 24 hours of ferulic acid effect. In a sample with T. taiwanensis was formed 61 mg/l of vanillic acid and 32 mg/l of 4-vinylguaiacol after 48 hours of ferulic acid effect, and in a sample of R. xylanophilus was formed 56 mg/l of 4-vinylguaiacol. In the sample with H. neptunia after 24 hours of ferulic acid effect was formed 296 mg/l of vanillic acid, which was up to 59% of the conversion of the added ferulic acid and in a sample with H. organivorans after 24 hours of ferulic acid effect was formed 71 mg/l of vanillic acid. However, after 48 hours of ferulic acid effect, vanillic acid degraded. In addition to screening of biotransformation potential, the sequence of bacterial enzymes, involved in biotransformation, was also compared with protein sequences in the database using the BLAST search tool. The presence of genes encoding enzymes involved in biotransformation has been demonstrated for almost all used microorganisms except H. neptunia, for which no gene has been identified. The highest number of genes present in bacteria was with the enzymes feruloyl-CoA synthetase, enoyl-CoA hydratase/isomerase, acetoacetyl-CoA thiolase and vanillin dehydrogenase.
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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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