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Isolation of PHA producing bacteria from mixed microbial consortia
Plachý, Petr ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is detection of polyhydroxyalcanoates (PHA) producing bacteria from activated sludge and effort for isolation of these bacteria. The theoretical part deals with general issues of PHA and of bacterial production of PHA. Also there is attention paid to characterization of activated sludge and to selected methods used in this thesis for detection of PHA producing bacteria. In the experimental part, mixed microbial culture of the activated sludge was cultivated on different carbon sources. Potential PHA producers was isolated from these cultures with the use of lipophilic staining with Nile red and phaC gene (essential for PHA synthesis) was detected with the use of polymerase chain reaction (PCR) in 11 of the isolated cultures. By DNA sequencing 8 bacterial cultures were identified. It was Klebsiella pneumoniae (1 x), Paenirhodobacter enshiensis (1 x) and Pseudomonas putida (6 x). Presence of PHA in biomass was detected in 2 of the 11 isolated cultures by Fourier transformation infrared spectroscopy (FTIR) analysis. The content of polyhydroxybutyrate (PHB) was determined with the use of gas chromatography to 9,33 % of dry biomass (Paenirhodobacter enshiensis) and to 1,18 % (unidentified culture).
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Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
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Production, characterization and application of polymers of malic and glutamic acids
Čangelová, Katarína ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Subject of this bachelor thesis was study of biopolymers production using microorganisms. Polymer of malic acid was produced by Aureobasidium pullulans, a yeast-like fungus. Biosynthesis of polymer of glutamic acid was carried out by Bacillus licheniformis bacterium. Particular polymers, their microbial producers and possible application in different areas are characterised in the theoretical part of the thesis. Through experiments, the effect of various conditions on cell growth and production of these polymers was studied. Cultivation conditions were optimised in terms of polymers production. Subsequently, waste substrates - whey and poultry feathers were used. Microbially-produced polymer of malic acid was hydrolysed with 2 M sulfuric acid. Concentration of malic acid was analysed by IEC with a conductivity detector. Concentration of polymer of glutamic acid was determined by a spectrophotometric method using CTAB.
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Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
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Utilization of lignocellulose materials for biotechnological production of polyhydroxyalkanoates
Kučera, Dan ; Márová, Ivana (referee) ; Obruča, Stanislav (advisor)
Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.
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Involvement of polyhydroxyalkanoates into stress response of bacteria
Kučera, Dan ; Skoumalová, Petra (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates (PHA) into stress response of bacteria. The theoretical part of the thesis deals with the possibility of establishing the viability of microorganisms using modern techniques, in particular flow cytometry. Furthermore, the research focused on selected stress factors and PHA involvement in stress response was described. In the experimental part of the work the stress response with regard to the ability to accumulate PHA was assessed. Bacteria Cupravidus necator was used for the experiment. Its ability to accumulate PHA at a later stage of growth increased resistance to ethanol, high temperature and freezing. Conversely, the PHA-producing strain showed lower resistance to the action of inorganic acids and bases. This may be caused by different morphology of PHA-producing cells. One of partial objectives was also to study the possibilities of staining of living cells accumulating PHA using Nile red. The research proved that the dye penetrates into living cells at elevated temperature of 40-45°C. This temperature is not lethal to the cells and the intensity of staining is sufficient to distinguish PHA-producing cells using flow cytometry; that can be applied in the selection of industrial PHA producers.
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