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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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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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Využití bioaditiv v produkci bazalky (Ocimum basilicum)
Pečenka, Jakub
This diploma work is focused on the use of bioadditives (products based on algae and bacteria) in the production of basil (Ocimum basilicum). In the literar rewiew, this bioadditives are described generally in terms of their material composition, effect and use in practise. The experimental part deals with a field experiment with basil, which took place in Lednice in the land of the Faculty of Horticulture, Mendel University in Brno in 2013. Basil was divided into 5 variants (4 + control). Individual variants were treated by Amalgerol Premium, EM -- farming Super aktiviert, Pro Milieu Terra and product from Rawat without trade name. The effect of these bioadditives on yield and nutritional parameters of basil were observed. Nutritional parameters were determined from fresh basil matter and from the dried matter. It was the total antioxidant capacity, content of phenols, flavonoids and dry matter. The results show that the application of these products has both positive and negative impact on the monitored parameters in basil. Yield parameters of these preparations were not significantly affected. The greatest effect has Amalgerol Premium and EM -- farming Super aktiviert on increase of total antioxidant capacity. EM -- farming variant was also found to increase total phenols and flavonoids.
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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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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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Využití bioaditiv v produkci bazalky (Ocimum basilicum)
Pečenka, Jakub
This diploma work is focused on the use of bioadditives (products based on algae and bacteria) in the production of basil (Ocimum basilicum). In the literar rewiew, this bioadditives are described generally in terms of their material composition, effect and use in practise. The experimental part deals with a field experiment with basil, which took place in Lednice in the land of the Faculty of Horticulture, Mendel University in Brno in 2013. Basil was divided into 5 variants (4 + control). Individual variants were treated by Amalgerol Premium, EM -- farming Super aktiviert, Pro Milieu Terra and product from Rawat without trade name. The effect of these bioadditives on yield and nutritional parameters of basil were observed. Nutritional parameters were determined from fresh basil matter and from the dried matter. It was the total antioxidant capacity, content of phenols, flavonoids and dry matter. The results show that the application of these products has both positive and negative impact on the monitored parameters in basil. Yield parameters of these preparations were not significantly affected. The greatest effect has Amalgerol Premium and EM -- farming Super aktiviert on increase of total antioxidant capacity. EM -- farming variant was also found to increase total phenols and flavonoids.
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