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Biotransformation of phenolic compounds by bacterium Schlegelella thermodepolymerans
Opial, Tomáš ; Dvořák,, Pavel (referee) ; Obruča, Stanislav (advisor)
The diploma thesis deals with the optimization of the biotransformation of ferulic acid into sensory active substances and production of P3HB by bacterium Schlegelella thermodepolymerans. Cultivation experiments were performed in a medium with various concentrations of ferulic acid. Four bacterial strains were used for these experiments. Attention was paid to the model strain DSM 15344, in which a bioinformatic analysis of genes encoding enzymes, that catalyzed the biotransformation reactions of ferulic acid was also performed. Ferulic acid metabolites, specifically sensory active substances such as vanillyl alcohol, vanillic acid, vanillin and 4-vinylguaiacol, were measured by HPLC. S. thermodepolymerans DSM 15344 exhibited a very fast utilization of ferulic acid already within 8 hours of cultivation. The highest yields of ferulic acid metabolites were detected between 8 and 24 hours depending on the initial concentration of ferulic acid. The main metabolites of ferulic acid were vanillic acid and vanillyl alcohol. Vanillic acid reached the highest concentration in 16 hours in the presence of ferulic acid at initial concentration of 0.5 g/l. The highest yield of vanillyl alcohol occurred in 8 hours with ferulic acid at initial concentration of 0.75 g/l. In addition to biotransformation to ferulic acid, the amount of produced P3HB was also measured by GC-FID. Schlegelella thermodepolymerans DSM 15344 produced the highest amount of P3HB at 72 h at the lowest initial concentration of ferulic acid (0.25 g/l). Within the bioinformatic analysis, the presence of one fcs gene was confirmed using the available databases and the BLAST tool, but this was not enough to construct the complete metabolic pathway of ferulic acid by the bacterium S. thermodepolymerans DSM 15344.
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Utilization of physico-chemical and spectroscopical methods in study on microbial extremofiles
Dobešová, Kateřina ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis is the determination od polyhydroxyalkanoates in the cells of extremophille bacterie, preformed by several methods. The chosen bacterium was Schlegelella thermodepolymerans DSM 15 344 Thermogravimetric analysis, infrared spectroscopy with Fourier transformation, and Raman spectroscopy were used in this thesis. The TGA method was used for the determination of extracellular and intracellular water in samples. The FTIR method was used in order to observe the amount od PHA, but aslo to observe the physical properties of the polymer in the sample, specifically to determine either amorphous or crystalline featuers of this polymer. Raman spectroscopy was used in oder to confirm the presence of PHA in the samples. All results e´were compared eith the results accomplished by gas chromatografy.
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Use of mealworm (Tenebrio molitor) for isolation of polyhydroxyalkanoates from bacterial biomass and simultaneous production of food
Najbrtová, Johana ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the use of mealworms (Tenebrio molitor) to isolate polyhydroxyalkanoates from bacterial biomass and current food production. Cupravidus necator H16 and Schlegelella thermodepolymerans were used to prepare bacterial biomass containing polyhydroxyalkanoates (PHA). The ability of mealworms to isolate PHA from bacterial biomass of various types was determined. First, the content of PHA and crude proteins in individual types of biomasses was determined. Subsequently, two biological isolation experiments were performed, in both cases the results were compared with a control sample that consumed bran. The conversion of feed into the form of fecal pellets was monitored, where the best results were obtained from a sample of mealworms fed with dried biomass C. necator H16. Furthermore, the weight loss during the experiment, the percentage of survivors, the amount of feed not consumed, and the number of pupae were evaluated. The fecal pellets obtained by biological isolation had a low PHA content and were therefore subjected to purification. The reagent with the best results was 0,1 M NaOH. Another part of this work was to determine the crude protein content of mealworm flour made from mealworms from experiment two. The sample fed with biomass produced by Schlegelella thermodepolymerans had the highest protein content.
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Novel approaches for PHA isolation from bacterial biomass
Dlouhá, Karolína ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The aim of this diploma thesis was study of the isolation of polyhydroxyalkanoates using a commercial surfactant and selected biosurfactants, which were sophorolipids, coconut soap, lecithin, lauryl glucoside, coco glucoside and cocamidopropyl betaine. PHA was isolated from Schlegelella thermodepolymerans DSM 15344, where the amount of NaCl in the production medium was first optimized. The molecular weight of the isolated polymer was analyzed by SEC-MALS. The largest structural changes were recorded for coconut soap. Possible impurities in the isolated polymer were analyzed by infrared spectrometry (ATR-FTIR). The least protein contamination of the polymer was recorded with coconut soap. From the above biosurfactants, coconut soap, lauryl glucoside and coco glucoside were selected, because the highest purity of PHA was obtained. However, coconut soap had the greatest potential. The isolation temperature and surfactant concentration were optimized for selected biosurfactants. The best results were obtained at 90 °C and a concentration of 5 g/L. Furthermore, the versatility of the isolation method was tested using selected biosurfactants on various thermophilic microorganisms, which were Chelatococcus composti DSM 101465, Schlegelella thermodepolymerans DSM 15264, Tepidimonas taiwanensis LMG 22826 and Aneurinibacillus thermoaerophilus H1.
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Production of polyesters by extremophilic bacteria
Bednárová, Kristína ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of the production of polyhydroxyalkanoates using thermophilic bacterial strains Schlegelella thermodepolymerans, Schlegelella aquatica and Thermomonas hydrothermalis. The aim of the thesis is to optimize the conditions of polymer production. In bacterial strains of the genus Schlegelella, phaC gene at the genotype level was confirmed. The ability to produce polyhydroxyalkanoates in the bacterial strain Thermomonas hydrothermalis at the phenotype level was demonstrated. The utilization of selected carbon sources was studied experimentaly. The production of PHA was optimized by selecting the appropriate substrate and cultivation temperature. The effect of precursors on the production of copolymers P(3HB-co-4HB) and P(3HB-co-3HV) was observed. The most promising carbon substrate for bacterial strain Schlegelella aquatica was glycerol while the best metabolic activity was at 45 °C. Strain Thermomonas hydrothermalis was able to produce the copolymer P(3HB-co-4HB) with extreme high molar fraction of 4HB. Optimal cultivation temperature was 55 °C. The highest yield of P(3HB) among selected bacteria, synthesized Schlegelella thermodepolymerans cultivated on xylose at 55 °C. For this strain, the effect of aeration on biomass growth and PHA production was observed. In an effort to reduce production costs, Schlegelella thermodepolymerans was cultivated on model hydrolysates of lignocellulose-rich materials. Finally, batch cultivation in a larger production volume in laboratory fermentor was performed.
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Biodegradation of bioplasts in compost environment
Vodička, Juraj ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This master’s thesis focuses on biodegradation of polyhydroxybutyrate (PHB) and polylactic acid (PLA). The theoretical part discusses an origin, properties and applications of investigated biopolyesters, and so the enzymology of their biodegradation. The experimental part deals with biodegradation of these polymers in liquid medium using several pure thermophilic bacteial strains and controlled composting of these bioplastics. Amongst six tested thermophilic bacterial strains only one showed PHB-biodegradation activity – strain Schlegelella thermodepolymerans. No degradation degree of amorphous or semi-crystalline PLA was observed. Mainly disintegration of both forms of PLA articles was observed in compost environment, thus the abiotic mechanism of its decomposition was indicated. After 4 weeks of composting, the relative weight loss of 99 % and 63 % was detected in amorphous and semi-crystalline PLA respectively. On the contrary, the weight loss of PHB after 4 weeks of composting reached 36 %, moreover, a half decrease of molar mass was observed using SEC. The surface erosive mechanism of PHB-biodegradation was stated using SEM. By monitoring of esterase, lipase and protease activities, no influence on the compost by polymer presence was concluded at statistical significance.
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