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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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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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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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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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Employment of thermophilic bacteria for biodegradation of synthetic and natural polymers
Csölle, Eduard ; Sedlář, Karel (referee) ; Obruča, Stanislav (advisor)
This bachelor’s thesis deals with the employment of thermophilic bacterium Schlegelella thermodepolymerans for biodegradation of selected polymers, namely poly(3-hydroxybutyrate) (PHB), polylactic acid (PLA), and poly(butylene adipate-co-terephthalate) (PBAT). The theoretical part focuses on the properties and origin of these bioplastics, the characteristics of the studied bacteria, and the general course of the biodegradation process. The experimental part discusses the twenty-day cultivation of three tested strains in the presence of the mentioned polymers and the subsequent evaluation of the degradation rate of these materials. The greatest increase in biomass content was observed by spectrophotometry for strain DSM 15344 when cultured on PHB. With blank samples (without polymers), it was shown that for significant growth, the bacteria had to truly utilize the polymers present. Gravimetric analysis of biomass content confirmed that PHB was the most suitable substrate for cultivation. The highest mass losses were recorded for PHB and were around 30 % across all three strains. For strain LMG 21645, the mass has decreased by almost 33 %. The weight losses of PLA and PBAT were considerably lower. Nevertheless, the most significant molecular weight loss was observed by SEC-MALS for PLA, reaching approximately 80 % for strain DSM 15264. SEM analysis of the polymer surface confirmed that the biodegradation of PHB occurs by enzymatic surface erosion. In contrast, the PLA and PBAT samples did not show any signs of biodegradation.
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Involvement of polyhydroxyalkanoates in stress response of thermophilic bacterium Schlegelella thermodepolymerans
Berger, Matyáš ; Nováčková, Ivana (referee) ; Slaninová, Eva (advisor)
This bachelor's thesis is focused on investigating the effect of different stress factors on the bacteria Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates. In this case, in the form of poly(3-hydroxybutyrate). The selected stress factors were temperature shock, repeated freezing and defrosting. The last stress factor was osmotic stress, caused by sodium chloride solutions of different concentrations. In the theoretical part, polyhydroxyalkanoates, thermophilic organisms, mechanisms of individual stresses and methods of analysis of polyhydroxyalkanoates were characterized. In the experimental part, two strains of the thermophilic bacteria Schlegelella thermodepolymerans were compared, namely M 15344 and DSM 15344. The stress resistance of both strains was compared by measuring viability using flow cytometry. During the measurement, the increase in optical density was monitored, and the content of P(3HB) in the biomass was determined using GC-FID. Morphological changes caused by stresses were observed using Cryo-SEM and STEM electron microscopy methods. The DSM 15344 strain showed better growth and P(3HB) production than the M 15344 strain, which, on the other hand, showed better stress resistance.
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Study on metabolic properties of the bacterium Schlegelella thermodepolymerans with respect to biosynthesis of polyhydroxyalkanoates
Chvátalová, Viktorie ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This bachelor's thesis deals with the metabolic properties of the thermophilic bacterium Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates (PHA). The aim of the study was to understand the metabolism and utilization of different carbohydrates (xylose, cellobiose, glucose and their combinations) depending on the cultivation time. In the experimental part, two bacterial strains (M15344 and DSM15344) were used. The increase in optical density was monitored during the measurements for the bacteria. Furthermore, the content of P(3HB) from the biomass was determined using GC-FID and the molecular weight of the polymer was determined using SEC- MALS analysis. By measuring residual sugar using HPLC-RI, the course of carbohydrate utilization was determined as a function of time. The results of both bacteria were compared and evaluated. DSM15344 showed better results in P(3HB) production than M15344, and cellobiose and xylose appeared to be the most suitable substrates for P(3HB) production.
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Production of polyhydroxyalkanoates by bacterium Schlegelella thermodepolymerans
Černý, Vojtěch ; Černayová, Diana (referee) ; Pernicová, Iva (advisor)
The aim of this work was to study capability of various strains of thermophilic bacteria Schlegelella thermodepolymerans incorporate diverse monomer units to structure of polyhydroxyalkanoates (PHA). Precursors for production mcl-PHA, 4-hydroxybutyrate and 3-hydroxyvalerate were tested. The theoretical part deals with characteristic of studied polymers, properties and applications and usage of thermophilic bacteria in industry. In experimental part there were undergone cultivations with different precursors, which were added in different times and concentrations. Four bacterial strains S. thermodepolymerans from various public collections of microorganisms were used during these cultivations. Levulinic acid, added at start of cultivation, was chosen as the best precursor. Afterwards there was cultivation with concentration line to find out, whether concentration influence composition of copolymer. Increasing the amount of precursor led to decreasing quantity of biomass and PHA, but there were very interesting materials with high amount of 3HV. Addition 8 g/l of precursor led up to 52 %. Subsequently there was found out molecular height of polymers by size-excluion chromatography (SEC) combined with multi-angle light scattering (MALS).
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Study on metabolism of polyhydroxyalkanoates in selected thermophilic bacteria
Reinohová, Nikola ; Sedláček, Petr (referee) ; Pernicová, Iva (advisor)
This thesis is focused on adaptive evolutionary engineering in the thermophilic bacterium Schlegelella thermodepolymerans M15344. The theoretical part deals with extremophilic microorganisms, biotechnological applications as well as polyhydroxyalkanoates and evolutionary engineering. In this work, adaptation of this microorganism to 40 °C and 60 °C was carried out using glucose and xylose substrates. Furthermore, repeated-batch cultivation at 50 °C on glucose and xylose was performed. In these experiments, the concentration of PHA produced in the biomass was determined by gas chromatography, and the concentration of residual sugars in the medium was determined by a method using DNS. Production of polyhydroxyalkanoates in the adapted strains from each experiment was compared with the non-adapted strain. Subsequently, the PHA produced by individual bacteria was studied using the SEC-MALS method, where the effect of adaptation on molecular weight was observed. The highest producing bacteria was adapted to 40 °C and the substrate was glucose. In this case, the biomass value was 6,74 g/l with a PHA percentage of 56.5 wt%. The bacteria was also adapted to 60 °C where it was able to grow and produce unlike the original bacteria.
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