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Determination of vibality of rhizobacterial cultures
Svobodová, Lucie ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on the study of viability of plant growth promoting rhizobacteria (PGPR). Viability was determined in three strains of Azotobacter vinelandii, namely CCM 289, DSM 87 and DSM 720, using flow cytometry with fluorescent probe PI, SYTOXTM Blue and DAPI after 120 and 72 hours of cultivation. Optimization of the appropriate fluorescent probe for the strain was performed, with the PI probe for strain CCM 289 being the most suitable. PI and SYTOXTM Blue probes can be used for strains DSM 87 and DSM 720. For the following experiments, strain DSM 87 was selected and subjected to the influence of different crosslinking reagents. Using a flow cytometer and staining with a fluorescent PI probe, the viability was verified after application of calcium chloride, barium, copper, ferric, aluminium and calcium sulphate solutions of 2, 0.2 and 0.02 wt. % to the culture. Calcium chloride, barium and calcium sulfate solutions had no significant effect on cell viability. On the other hand, when ferric chloride was used, a trend was observed where dead cells decreased with decreasing concentration of the solutions. This effect was also achieved with aluminium and copper chloride, but the use of the most concentrated solution resulted in the inactivation of a greater number of cells than in the previous case, whereas aluminium chloride resulted in the loss of viability of most of the cells present. Viability was also verified for cells released from the prepared gels. For the experiments, solutions of the aforementioned crosslinking agents were chosen at a concentration of 2 wt.%, and the culture was subjected to gelation under the experimentally determined conditions. A portion of the gels was subsequently left in phosphate buffer to allow for the re-release of cells. To facilitate this release, the enzyme alginase was added to break down the alginate. It was found that a concentration of 2 wt % of the selected crosslinking agents did not affect cell viability, i.e., the cells released from the gel appeared to be viable.
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Utilization of advanced techniques of electron microscopy in altrastructural analysis of microbial cells and microbial biotechnology products
Havlíčková, Anna ; Sedláček, Petr (referee) ; Krzyžánek,, Vladislav (advisor)
In the theoretical part, this thesis deals with direct and indirect mechanisms of plant growth-promoting rhizobacteria, which is followed by a section on the selected model organism Azotobacter vinelandii. The second part of the theoretical section deals with electron microscopy starting with the possible interactions of the electron beam with the sample, through types of electron microscopes to various methods of preparing biological samples. The experimental part discusses the cultivation of selected bacterial cultures, the gelation of the cell cultures using chosen cross-linking agents (calcium chloride, malic acid, glucono--lactone with calcium carbonate) and, above all, various methods of preparation and analysis of pure and encapsulated cultures by the means of electron microscopy. In particular, cryogenic scanning electron microscopy and scanning transmission electron microscopy imaging were selected. Using both techniques, poly-3-hydroxybutyrate granules, which were confirmed to be elastic even at cryogenic temperatures, were observed inside the bacteria. Furthermore, it was found that cross-linking of extracellular alginate occurs even in samples without the addition of cross-linking agents. However, the hydrogel network present in these samples was significantly thinner than in samples with the addition of all cross-linking agents used. In this work, the density of the hydrogel network cross-linked with glucono--lactone was also compared. When using this agent with the addition of calcium carbonate, a significantly denser hydrogel network was observed in comparison with using the agent alone. Last but not least, when comparing the obtained results with the literature, it was found that the A. vinelandii bacteria form capsules, during the formation of the capsules the shape of the cells changes from an oval to a spherical bacterium and also the size of the given cell is reduced. From the resulting images, it was found that these capsules are made up of two layers, to which literature refers as electron-transparent intine and electron-dense exine.
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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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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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Self-encapsulation of Plant Growth Promoting Rhizobacteria as a way towards new generation of bioinoculants
Gašparová, Dominika ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This bachelor thesis focuses on Plant Growth-Promoting Rhizobacteria that can be used as a suitable ecological alternative to conventional fertilizers. However, most of today's approaches to incorporating PGPR into the process of soil fertilization are convoluted and costly. Crosslinking of alginate self-produced by PGPR offers a new possibility of plantgrowth- promoting bacteria encapsulation. The aim of this thesis consists of preparing gel matrices with incorporated bacteria, followed by testing the bacteria's viability and quantity. Firstly, experiments were carried out to determine various properties of gels produced by the chosen representative bacteria Azotobacter vinelandii. These properties include the molecular weight of alginate (with the highest molecular weight of strain CCM 289 (329,78± 6,8) kDa), the alginate's amount in the gel, the content of PHB (34%-44% CDW), and the gel's overall rheological properties. The complex modulus (as a representation of rheological properties) of the firmest gel reached the value of 13,34 kPa. The bacteria content was examined by flow cytometry, the CFU method, and spectrophotometry. The viability of bacteria was determined by analyzing the fluorescence intensity via fluorescent dyes (propidium iodide, fluorescein diacetate, rhodamine 123, calcein AM), then by using the CFU method, and also via the plate method. Furthermore, the effects of various factors such as different pH levels (pH 3, 7, and 10) or adding the specific alginatedegrading enzyme alginase were also examined. Releasing of the bacteria was observed over the course of seven days using flow cytometry and the CFU method. The plate method proved the bacteria's survival after gelation and without any source of carbon. The largest disintegration of gels occurred in acidic pH. Moreover, the effect of alginase on crosslinked bacterial gels and synthetic alginate were very similar. The survival of bacterial cultures in gel and their continuous release are significant findings in the development of novel bioinoculants based on this new concept.
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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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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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Development and experimental evaluation of bioactivity of the next-generation bioinnoculants
Súkeník, Martin ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
This diploma thesis aimed to characterize specific mechanisms of biological activity in plant growth-promoting rhizobacteria (PGPR) commonly found in the rhizosphere of plants and to study the effects of liquid and gel application of PGPR bioinoculants on plants in a controlled environment. An original concept of self-encapsulation via cross-linking of alginate directly produced during their culturing was proposed for the preparation of gel bioinoculants based on these PGPRs. To validate the efficacy of this concept, all selected strains had to be subjected to gelation experiments with the crosslinking agent CaCl2 2 wt. % under alginate gel formation. Three specific strains of Azotobacter vinelandii DSM 87, DSM 720 and CCM 289 were selected for the preparation and characterization of the bioinoculants. The material properties of the formed gels were characterized by oscillatory rheometry (amplitude test). All strains were subjected to gravimetric determination of alginate and biomass concentrations. Flow cytometry with fluorescent probe PI, DAPI and SYTOX™ Green was used to verify cell viability immediately after culturing. Furthermore, the drying of gels was performed using a lyophilizer, where the lyophilized gel was used in swelling experiments followed by drying. To verify the biological activity of the bioinoculants, the ability of all strains to dissolve and utilize phosphate from the agar medium was qualitatively verified. Furthermore, the production of indole-3-acetic acid was spectrophotometrically verified for all strains. The production of siderophores was also verified qualitatively and quantitatively. Cultivation pilot experiments on the lettuce (Lactuca sativa) plant were performed with the addition of both liquid and gel carrier PGPR compositions; as a negative control, the plant was left without the addition of carrier PGPR composition. The results from the pilot growth experiment showed a positive growth effect of both gel and liquid PGPR composition on the plants. At the end of the growing experiment, higher leaf and root system lengths were recorded after the application of both forms of PGPR to the soil, specifically, the average leaf length with the addition of gel carrier PGPR composition was 4.3±1.0 cm and 4.3±1.2 cm for the gel carrier PGPR composition, and the average root system length was 18.3±5.2 cm and 17.4±6.0 cm, respectively. The dry weight per plant grown with the addition of liquid and gel PGPR was the same 0.19±0.07 g. The highest content of chlorophyll a, chlorophyll b and carotenoids in the leaves was spectrophotometrically detected in plants grown with the addition of liquid PGPR composition, specifically chlorophyll a was found to be 6±1 µg/ml, chlorophyll b 7±2 µg/ml and carotenoids 3.6±0.5 µg/ml. The diversity of soil microbiomes collected from the soil without the addition of PGPR composition, with the addition of liquid PGPR composition and with the addition of gel PGPR composition was analysed using BIOLOG EcoPlate™ plates. The diversity of the microbiome was evaluated using the average well colour development, i.e., the AWCD parameter, and the Shannon diversity index, H'. The highest utilization of the widest number of carbon sources on the plate was recorded for the soil microbiome with the addition of the gel PGPR composition. The aforementioned soil microbiome also showed the highest value of the H' index.
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Involvement of various gelation strategies in development of next-generation bioinnoculants
Krýslová, Jana ; Smilek, Jiří (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the investigation cross-linking approaches of the bacterium Azotobacter vinelandii, which belongs to the PGPR group and is capable of producing alginate extracellularly. This crosslinking concept represents a novel approach to development of bioinoculants based on the PGPR. The first part is dedicated to investigating the effect of oxygen on the gelation of bacterial cultures itself. Experimentally, it was found that bacterial cultures in a total volume of 50 and 100 ml in 250 ml Erlenmeyer flasks showed spontaneous gelation already during the cultivation process, while the formed gels were measured rheometrically and showed values of const. complex modulus 0,026 kPa and 0,030 kPa. With a bacterial culture in a total volume of 150 ml, spontaneous cross-linking itself did not occur. In addition to gelation testing, the content of PHB and extracellular alginate and its molecular weight were determined in the bacterial cultures using size exclusion chromatography with multi-angle light scattering (SEC-MALS) and the mechanical properties of the resulting gels using rheometry. Cross-linking of bacterial cultures took place mainly with the use of CaCl2 as a cross-linking agent, but various alternative methods of gelation were tested as well (by means of polyvalent ions, weak organic acids, precipitants such as DMSO, emulsion gelation). The effect of glucono-D-lactone (GDL) as a gelling agent based on a slow release of crosslinking ions from their insoluble form was also tested. The strongest gels were formed by the addition of polyvalent ions (they ranged in the value of the complex modulus of 10 kPa, in the case of the use of weak acids and DMSO, the gels ranged in the value of the complex modules in the range of 1 to 10 kPa. The addition of GDL did not create gels immediately, but delayed, and gelation was observed in time.The formed gel ranged in value up to 1 kPa.
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Self-encapsulation of Plant Growth Promoting Rhizobacteria as a way towards new generation of bioinoculants
Gašparová, Dominika ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
This bachelor thesis focuses on Plant Growth-Promoting Rhizobacteria that can be used as a suitable ecological alternative to conventional fertilizers. However, most of today's approaches to incorporating PGPR into the process of soil fertilization are convoluted and costly. Crosslinking of alginate self-produced by PGPR offers a new possibility of plantgrowth- promoting bacteria encapsulation. The aim of this thesis consists of preparing gel matrices with incorporated bacteria, followed by testing the bacteria's viability and quantity. Firstly, experiments were carried out to determine various properties of gels produced by the chosen representative bacteria Azotobacter vinelandii. These properties include the molecular weight of alginate (with the highest molecular weight of strain CCM 289 (329,78± 6,8) kDa), the alginate's amount in the gel, the content of PHB (34%-44% CDW), and the gel's overall rheological properties. The complex modulus (as a representation of rheological properties) of the firmest gel reached the value of 13,34 kPa. The bacteria content was examined by flow cytometry, the CFU method, and spectrophotometry. The viability of bacteria was determined by analyzing the fluorescence intensity via fluorescent dyes (propidium iodide, fluorescein diacetate, rhodamine 123, calcein AM), then by using the CFU method, and also via the plate method. Furthermore, the effects of various factors such as different pH levels (pH 3, 7, and 10) or adding the specific alginatedegrading enzyme alginase were also examined. Releasing of the bacteria was observed over the course of seven days using flow cytometry and the CFU method. The plate method proved the bacteria's survival after gelation and without any source of carbon. The largest disintegration of gels occurred in acidic pH. Moreover, the effect of alginase on crosslinked bacterial gels and synthetic alginate were very similar. The survival of bacterial cultures in gel and their continuous release are significant findings in the development of novel bioinoculants based on this new concept.
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