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Coproduction of polyhydroxyalkanoates and violacein employing members of the genus Janthinobacterium
Uhrová, Kateřina ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This bachelor's thesis deals with the study of appropriate conditions for the cultivation of bacteria of selected genera Janthinobacterium, specifically strains CCM 160 and CCM 1888 and the genus Iodobacter fluviatilis strain CCM 3308 from the Czech collection of microorganisms. The most suitable conditions for bacterial growth were investigated. The aim was to identify ideal production medium, cultivation volume, temperature and conditions in the form of submerged and static cultivation. At the same time, one of the strains used was chosed, which appeared to be the best producer of metabolites. Production of the purple pigment violacein and polyhydroxyalkanoates (PHA) was observed during the cultivations. Violacein was extracted using methanol and the concentration of the produced violacein content was measured using UV/VIS spectrophotometry. The PHA content was determined using gas chromatography. The obtained data were compared with each other and it was determined whether the production of both metabolites is mutally affected. Furthermore, cultivation on a solid carrier was carried out, which proved more effective in the production of violacein than in a liquid medium. This type of cultivation could in the future lead to the commercial production of violacein, which has a number of positive properties. The 16S rRNA and phaC genes were detected using the multiplex PCR method.
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Use of optical and microfluidic techniques for analysis and separation of microbial cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek,, Ota (advisor)
The presented diploma thesis primarily focuses on Raman spectroscopy and its connections to microfluidic techniques, along with potentially biotechnologically interesting products of bacterial strains Janthinobacterium lividum and Azotobacter vinelandii. The methods were chosen for their non-destructive nature, which allows for rapid cell analysis and comparison with gas chromatography. After analysis, cells can be further cultivated or a generation with excessive production of a selected metabolite can be prepared. Janthinobacterium lividum is a bacterial strain that produces the pigment violacein, which has significant biotechnological potential as a drug, antioxidant, fluorescent probe, or dye. The ability to distinguish between cells producing and not producing violacein can have wide applications in diagnosis and identification. In the experimental part, the spectra of three strains were measured, based on which Janthinobacterium lividum CCM 160 was selected for analysis using Raman tweezers, along with Cupriavidus necator H16. By evaluating with principal component analysis, the distinguishability of pigmented cultures from non-pigmented ones was determined, and a potential sorting marker of 1 140 cm-1 (corresponding to violacein) was selected for further measurements. The biotechnological potential of the Azotobacter vinelandii strain lies in its ability to fix nitrogen from the atmosphere, produce alginate, and polyhydroxybutyrate. The experimental part of the thesis focuses on identifying Raman spectra of five strains and the extracted alginates from them. Polyhydroxybutyrate (PHB) is a biodegradable polymer that replaces petrochemical plastics. For PHB production, Azotobacter vinelandii is an adept for commercial production, as it is one of the few that produces PHB directly from atmospheric nitrogen, which can lead to reduced production costs and thus higher representation of degradable plastics in the market. The highest response for PHB in Raman spectra was measured for Azotobacter vinelandii DSM 720 and the lowest for Azotobacter vinelandii DSM 85. Quantitative information for comparison was obtained from gas chromatography, which confirmed the measurements from Raman spectroscopy. By evaluating the measured Raman spectra of pure alginates, it is possible to assume that they are heteropolymeric fractions, as the spectra showed the expression of various monomers. Depending on the composition of subunits, the properties of alginate change, so it is necessary to know the composition for its adjustment for further use in medicine or the food industry. From the five Azotobacter vinelandii strains, producers with the highest and lowest PHB content were selected, which were subsequently analysed using Raman tweezers. By evaluating with principal component analysis, the distinguishability of these strains and their separability using the sorting parameter of 1 060 cm-1 was determined.
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