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Effect of culture medium composition on selected characteristics of microbial culture of Aneurinibacillus sp.
Sklárová, Viktória ; Hrabalová, Vendula (referee) ; Buchtíková, Iva (advisor)
The aim of this bachelor thesis was to identify the key components of the culture medium and to optimize the composition of the culture medium for the thermophilic strain Aneurinibacillus sp. AFN2. The theoretical part discusses extremophilic microorganisms, especially thermophilic ones, and their ability to produce polyhydroxyalkanoates, followed by a description of the structure, properties, synthesis, and applications of polyhydroxyalkanoates. In the experimental part of the work, cultivations were performed to optimize the production medium, inoculation ratio, inoculation culture time and the effect of medium cooling on the production of polyhydroxyalkanoates. Finally, a last cultivation was performed in which the initial and optimized cultivation conditions were compared. All cultivations were performed on mineral and complex production medium. After a series of experiments, the optimized conditions for the complex medium were determined to be the medium composition without NaCl addition, an inoculation ratio of 10%, an inoculation culture cultivation time of 24 hours. The optimum conditions of mineral medium were determined as follows, the composition of the mineral medium remained unchanged from the initial one, the optimum inoculation ratio was determined to be 15% and the inoculum culture cultivation time was established to be 24 h.
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Optimization of the cultivation media for thermophilic bacterium Caldimonas thermodepolymerans
Fajtová, Zuzana ; Černayová, Diana (referee) ; Obruča, Stanislav (advisor)
The subject of the bachelor ‘s thesis is the optimization of the cultivation conditions of the thermophilic bacterium Caldimonas thermodepolymerans. The theoretical part provides a general characterisation of PHA materials, their synthesis, degradation, and applications. In addition, extremophilic microorganisms and the bacterium C. thermodepolymerans are discussed in the theoretical part. The experimental part of the thesis focuses on the production of the PHB by the bacterium C. thermodepolymerans DSM 15344. A series of experiments were carried out to optimise the cultivation conditions. The influence of temperature, inoculum ratio, inoculum age, mineral medium composition, and concentrations of nitrogen sources in the mineral medium on culture growth and PHB production was investigated. It was found that the original conditions set were already close to optimal for the cultivation. A higher yield compared to the control conditions was observed with a combination of 0.5 g/l yeast extract and 1.5 g/l ammonium chloride, resulting in a 7% increase in PHB.
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Study on metabolism of thermophillic bacterium Caldimonas thermodepolymerans
Krempaská, Vladimíra ; Sedlář, Karel (referee) ; Buchtíková, Iva (advisor)
Polyhydroxyalkanoáty (PHA) sa radia medzi mikrobiálne polyestery a bunkám dokážu poskytnúť vnútrobunkové zásoby uhlíka a energie. Hlavná výhoda PHA spočíva v tom, že ide o biodegradovateľné a biokompatibilné polyméry. Ich fyzikálne vlastnosti sú porovnateľné s petrochemickými plastami, ktoré by mohli v budúcnosti čiastočne nahradiť. Avšak, ich biotechnologická produkcia je stále veľmi nákladná. Preto sa hľadajú rôzne alternatívne metódy, ktoré by dokázali znížiť tieto procesné výdavky a ako jedna z možností sa javí využitie extrémofilných mikroorganizmov. Za jedného z vhodných kandidátov na priemyselnú produkciu PHA sa považuje termofilná baktéria Caldimonas thermodepolymerans. K jej ďalším výhodám okrem iného patrí aj to, že dokáže spracovávať sacharidy, ktoré sú vo veľkej miere obsiahnuté v lignocelulózových odpadoch. Táto práca bola konkrétne zameraná na štúdium metabolizmu troch lignocelulózových sacharidov (xylózy, glukózy a celobiózy) a ich vzájomných kombinácií u zbierkového kmeňa DSM 15344. V experimentálnej časti práce boli realizované kultivácie na jednotlivých sacharidových substrátoch, kde následne zo získanej biomasy bol stanovený celkový obsah PHA pomocou CG-FID. Na záver bola vyhodnotená expresia vybraných génov xylF a gtsA pri zmenách sacharidových substrátov v priebehu kultivácie za využitia RT-qPCR. U študovaného zbierkového kmeňa DSM 15344 bola pozorovaná preferencia xylózy a celobiózy ako substrátu.
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Study of the production of polyhydroxyalkanoates using selected thermophilic representatives of the genus Aneurinibacillus
Řeháková, Veronika ; Kovalčík, Adriána (referee) ; Pernicová, Iva (advisor)
The subject of the diploma thesis is the production of PHA copolymers by thermophilic Gram-positive bacteria of the genus Aneurinibacillus. The theoretical part is devoted to the general characterization of PHA materials, their properties, use, and also to the production of PHA polymers by these bacteria. The experimental part deals with the production of PHA copolymers using selected thermophilic members of the genus Aneurinibacillus. Firstly, the specific enzyme activity of PHA synthases was determined, and then the production of PHA copolymers was screened using selected lactones (-valerolactone, -hexalactone, -valerolactone) and diols (1,6-hexanediol, 2,3- butanediol and 1,4-butanediol). These experiments were performed with six thermophilic producers by the members of the genus Aneurinibacillus, which were isolated from compost and activated sludge. Tested microbial strains have demonstrated the ability to integrate interesting monomers into the PHA structure, including 4-hydroxyvalerate (represented up to 69.3% of the total PHA), 5-hydroxyvalerate (up to 47.1%), or 4-hydroxyhexanoate (up to 31.9%). Subsequently, a closer characterization of the obtained PHA (which were gained by the production of the best producers) using advanced methods (DSC, SEC-MALS, FT-IR) was performed. Finally, the screening of the PHA copolymers production was performed in laboratory bioreactors.
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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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Thermophilic microorganisms application to biodegradation
Varmužová, Tamara ; Márová, Ivana (referee) ; Babák, Libor (advisor)
This thesis is focused on study of biodegradability of modified polyurethane elastomeric films in synthetic medium with minerals and vitamins on tempered shaker by mixed thermophilic aerobic bacterial culture Bacillus and Thermus genera. In most cases addition of all used fillers (carboxymethyl cellulose, hydroxyethyl cellulose, acetylated cellulose, acetylated starch and glutein) led to increased biodegradability of elastomeric films with modifying agent in comparison with elastomeric films without modifying agent (referential). The growth of cultures was strongly increased in presence of elastomeric films modified by 10 % acetylated cellulose and 10 % carboxymethyl cellulose. Elastomeric film biodegradation mechanism included probably two stages: abiotic destruction of elastomeric films and consequent utilization of degradation products by bacterial culture.
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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
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Identification and isolation of PHA producing bacteria
Pernicová, Iva ; Ondrejovič, Miroslav (referee) ; Rychtera, Mojmír (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.
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Complementary analysis of procaryotic cells by electron microscopy and Raman spectroscopy
Ikrényiová, Terézia ; Sedláček, Petr (referee) ; Hrubanová, Kamila (advisor)
This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.
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production of PHA by selected extremophilic bacteria
Šarköziová, Patrícia ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor´s thesis deals with production of polyhydroxyalkanoates (PHA) by thermophilic bacterial strains of Chelatococcus composti and Chelatococcus sambhunathii. We were finding out the best conditions for production of PHA granules inside the cells. In the beginning of the experiment we employed PCR to detect the phaC gene, encoding for PHA synthase, which is requisite for production of PHA. We also analyse the best source of carbon, optimal temperature for production of polyhydroxyalkanoates and influence of chosen precursors to grow of biomass and production of copolymers. The last part of this experimental thesis deals with research utilization of selected model hydrolysates. Glucose was the best source of carbon for both bacteria Chelatococcus composti and Chelatococcus sambhunathii and optimal temperature for grow and production of P(3HB) was 45°C. The applied precursors inhibited cell grow, but both bacterial strains Chelatococcus composti and Chelatococcus sambhunathii could produce copolymer P(3HB-co-3HV) after additiong of precursors sodium propionate and valeric acid. For both bacterial strains was the most useful model hydrolysates, which contains the biggest part of glucose. Morever, bacterial cells containing PHA granules were observed by fluorescence and transmission electron microscopy.
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