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Hydrogel carriers of microorganisms for modern environmental aplications.
Súkeník, Martin ; Slaninová, Eva (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with the optimization of cultivation and gelation procedures by bacterium Azotobacter vinelandii, which belongs to a PGPR group, capable of synthesis of intracellular polyhydroxyalkanoates and extracellular alginate. The alginate produced by the bacteria is crosslinked with a calcium chloride solution to form a hydrogel carrier containing the described bacterium. This unconventional concept simplifies bacterial encapsulation and production of hydrogel carriers, furthermore expands its usability in modern environmental applications. In the first part of this thesis, three strains (DSM 85, 87, 720) were selected to measure a concentration of produced alginate, its molecular weight was determined by a SEC-MALS technique and M/G ratio was analysed by infrared spectroscopy. The Azotobacter vinelandii strain DSM 87 reached the highest concentration of the alginate (4.9 ± 0.6) g/l by gravimetric determination on the 5th day of cultivation. The concentration of produced PHB ranges from 48 to 6 % of CDW, which was determined by gas chromatography. Nevertheless, the strain DSM 720 showed the best gelation properties and was used in the second part of this thesis for further optimization of the cultivation process, and for the analysis the formation of gelation procedure under different temperatures, gelation time and concentrations of crosslinking solution. The formed gels were compared by rheological measurements of amplitude and frequency tests.
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Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.
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Study of polyhydroxybutyrate production in bacteria
Melušová, Soňa ; Babák, Libor (referee) ; Márová, Ivana (advisor)
Presented work is focused on study of polyhydroxybutyrate production in bacteria. In theoretical part short characterization of PHB was given and the most common representative of wide group of polyhydroxyalkanoates (PHA) were described. Then, production of PHB and copolymer P(HB-co-HV) in selected bacterial strains was experimentally proven. First, PHB production in Bacillus megaterium using synthetic medium was studied. The PHB content in cells was increased during cultivation under limiting conditions, despite low growth. Addition of ethanol into production media resulted in increased PHB synthesis as well as biomass production (21 % PHB of 1,8 g/l biomass). Further, BM medium containing 8 g/l glucose was tested. PHB production was more than 1 g/l at significant growth increase when compared with synthetic medium. The bacteria B.megaterium showed, except glucose, ability to utilize maltose and xylose. Another cultivations were tested with bacterial strain Azotobacter vinelandii, which is capable of copolymer P(HB-co-HV) synthesis. Maximal growth and copolymer content was reached on Burk's medium with 30 g/l of glucose. Addition of peroxide to growth medium influenced P(HB-co-HV) synthesis to 46 % of 2,6 g/l biomass. Bacteria A.vinelandii showed the best growth on maltose, even compared with glucose (54 % copolymer of biomass content). Finally, PHB production on industrial waste product – whey was monitored. Using Plackett-Burman design for statistical media optimization, the whey content was modified. B.megaterium grown on adjusted whey reached 0,5 g/l PHB, 32 % of cell's content.
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