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Bioinformatics study of the third generation sequencing platforms applied on a thermophile
Umair, Mohammad ; Řeháková, Veronika ; Buchtikova, Iva ; Bezdicek, Matej ; Obruca, Stanislav ; Sedlář, Karel
This study compares the efficiency of Pacific Biosciences technology (PacBio) and Oxford Nanopore Technology (ONT) in sequencing, assembling, and annotating the Aneurinibacillus sp AFn2 bacterium. We aim to evaluate the performance based on contiguity, depth, and functional annotation of the resulting genome. Using ONT we generated 152,047 long reads assembling into 2 contigs with total base count of 0.4 billion which provided us efficient assembly while PacBio produced 139,701 reads, assembling into 21 contigs with a total base count of 1.4 billion. Functional annotation revealed differences in the number of coding sequences, with PacBio detecting more comprehensive gene sets than ONT. The comparative analysis done in this research shows the strengths and limitations of both the platforms, with ONT providing higher assembly contiguity and PacBio offering greater detail in genetic content. We aim to offer insights of both the sequencing technologies, guiding researchers in selecting the appropriate technology.
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Biosynthesis of polyhyroxyalkanoates in non-suflur purple bacteria
Fiala, Tomáš ; Vodička, Juraj (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis focuses on polyhydroxyalkanoates (PHA) and their production using non-sulfur purple bacteria, specifically Rhodospirillum rubrum. The cultivation conditions were optimized especially with respect to the determination of NaCl concentrations for the following evolution experiments. Then this microorganism was repeatedly cultured at a high concentration of NaCl (40 g/L). Passage system was used in this cultivation with 48 h between inoculations under aerobic conditions in the dark. In the samples cultivated in this way, the growth rate was investigated using optical density and the amount of biomass and PHA using gas chromatography with an FID detector. As the last experiment PCR analysis was performed to test the 16S rRNA and phaC genes. The conclusion of the work is the successful adaptation of R. rubrum to a concentration of 40 g/l NaCl. Furthermore, the positive effect of stress stimulus on the increased PHA production was proven.
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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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Determination of vibality of rhizobacterial cultures
Svobodová, Lucie ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on the study of viability of plant growth promoting rhizobacteria (PGPR). Viability was determined in three strains of Azotobacter vinelandii, namely CCM 289, DSM 87 and DSM 720, using flow cytometry with fluorescent probe PI, SYTOXTM Blue and DAPI after 120 and 72 hours of cultivation. Optimization of the appropriate fluorescent probe for the strain was performed, with the PI probe for strain CCM 289 being the most suitable. PI and SYTOXTM Blue probes can be used for strains DSM 87 and DSM 720. For the following experiments, strain DSM 87 was selected and subjected to the influence of different crosslinking reagents. Using a flow cytometer and staining with a fluorescent PI probe, the viability was verified after application of calcium chloride, barium, copper, ferric, aluminium and calcium sulphate solutions of 2, 0.2 and 0.02 wt. % to the culture. Calcium chloride, barium and calcium sulfate solutions had no significant effect on cell viability. On the other hand, when ferric chloride was used, a trend was observed where dead cells decreased with decreasing concentration of the solutions. This effect was also achieved with aluminium and copper chloride, but the use of the most concentrated solution resulted in the inactivation of a greater number of cells than in the previous case, whereas aluminium chloride resulted in the loss of viability of most of the cells present. Viability was also verified for cells released from the prepared gels. For the experiments, solutions of the aforementioned crosslinking agents were chosen at a concentration of 2 wt.%, and the culture was subjected to gelation under the experimentally determined conditions. A portion of the gels was subsequently left in phosphate buffer to allow for the re-release of cells. To facilitate this release, the enzyme alginase was added to break down the alginate. It was found that a concentration of 2 wt % of the selected crosslinking agents did not affect cell viability, i.e., the cells released from the gel appeared to be viable.
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Bioinformatic analysis of extremophillic bacteria with respect to their capability of polyester accumulation
Bočková, Sabina ; Sedlář, Karel (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on the bioinformatic analysis of extremophilic bacteria in the context of their ability to accumulate Polyhydroxyalkanoates (PHA) – microbial storage polyesters. Specifically, we conducted bioinformatic research using the online available databases BacDive and BLAST to identify new thermophilic and psychrophilic bacterial strains with genetic predispositions for PHA synthesis. The identification of these potential PHA producers was based on the presence of the phaC gene (using the phaC gene from the bacterium Cupriavidus necator as a template). Using the BLAST database, the optimal cultivation temperature was then verified for the selected candidates in the BacDive databse. Selected bacterial strains that contained the phaC gene and fit into the group of themophiles or psychrophiles wre thoroughly characterized through literature review. Based in the obtained data, two tables were cimpiled containing a total of 60 themophilic and 60 psychrophilic bacteria with genetic predispositions for the PHA synthesis. In the experimental part, five psychrophilic bacteria of the genus Massilia were selected and the presence of the phaC gene was exymined using polymerase chain reaction (PCR). The presence of the class I phaC synthase was confirmed in two other bacteria. In one strain, it was likely that DNA isolation in the required purity and quantity was not successful.
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Biotechnological production of polyhydroxyalkanoates by thermophiles
Kouřilová, Xenie ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHAs) are microbial biopolymers that may provide a partial alternative to petrochemically produced plastics. Their main advantages are biodegradability, biocompatibility, and the possibility of production from renewable sources. However, the cost of their production is still higher than that of petroleum-based polymers. To increase the competitiveness of biotechnological processes, the concept of Next Generation Industrial Biotechnology (NGIB) has been introduced by other authors. This concept relies on the use of extremophilic microorganisms. When extremophiles are implemented in fermentation processes, the robustness of these technologies is increased and at the same time sterility requirements can be reduced. In the context of PHA production using extremophiles, a subset of halophilic microorganisms is relatively well mapped. Significantly less information is available on another interesting category, namely thermophiles. In line with the idea of NGIB, it is the production of PHAs by thermophilic bacteria that is the focus of this thesis. Attention is paid mainly to the genera Caldimonas, Rubrobacter, and Tepidimonas. For several tested representatives of these genera, the parameters investigated were the optimal cultivation temperature, suitable carbon substrate and the ability to produce copolymers. Based on this basic screening parameters, the most promising producers were selected and subjected to further experiments. Representatives of the genus Rubrobacter have the advantage of being gram-positive non-sporulating bacteria, as the risk of contamination of the isolated polymer by pyrogenic lipopolysaccharides present in the cell wall of gram-negative microorganisms is eliminated. The bacterial strain Tepidimonas taiwanensis LMG 22826T was able to produce high yields of biomass and PHA on a mixture of glucose and fructose substrate. Using grape pomace extract, almost identical values to those obtained on pure substrates were achieved. The thermophile Caldimonas thermodepolymerans DSM 15344, originally named Schlegelella thermodepolymerans, is a very promising PHA producer on xylose-based substrates. These substrates can be, for example, hydrolysates of lignocellulosic materials, which represent a sustainable source of carbon. Their suitability for the cultivation of C. thermodepolymerans was tested on model hydrolysates composed of pure carbohydrates. To approximate real samples, the effect of potential microbial inhibitors present in lignocellulosic hydrolysates was also investigated. An innovative isolation protocol for the recovery of PHAs based on osmotic stressing of thermophilic and halophilic microorganisms under elevated temperature with the addition of a low concentration surfactant solution was also developed as part of the thesis.
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Biotechnological production of polyhydroxyalkanoates employing extremophilic purple bacteria
Rubanová, Blanka ; Samek, Ota (referee) ; Obruča, Stanislav (advisor)
The presented thesis focuses on studying the metabolism of polyhydroxyalkanoates (PHAs) in extremophilic purple bacteria, specifically Rhodoblastus acidophilus and Rhodobaca barguzinensis. The bacteria's ability to synthesize PHAs was examined in various media and using different carbon sources. Initially, optimal conditions including media volume, light presence, cultivation duration, and inoculum size were determined for the most suitable bacterial growth. Subsequently, the PHA content in bacteria cultivated in different media and with various carbon sources was analysed using gas chromatography. For a better characterization of bacterial metabolism and morphology, scanning transmission electron microscopy (STEM) and Fourier-transform infrared spectroscopy (FTIR) were used. The bacteria's ability to synthesize PHA was also examined at the genotype level using polymerase chain reaction (PCR) method. The presence of the phaC gene, encoding PHA synthase class I, was detected in bacterial DNA. Additionally, an experiment was conducted to cultivate bacteria under conditions favourable for the synthesis of microbial pigments (carotenoids and bacteriochlorophyll a). The absorption spectrum of extracted pigments was measured using UV-VIS spectroscopy.
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Preparation and characterization of lignin modified by laccase enzyme
Hrušková, Lucie ; Obruča, Stanislav (referee) ; Kalina, Michal (advisor)
This master’s thesis focuses on the enzymatic modification of commercially available lignin using laccase enzyme and monitoring its impact on selected physical and chemical properties. Lignin is an attractive material for the production of renewable chemicals, materials, and energy, but its complex chemical structure poses a major obstacle to its effective utilization. Enzymatic modification appears to be a promising solution for its processing. Experiments were conducted under various conditions, such as pH, enzyme dosage, and reaction time. In the subsequent part of the experiment, the modified lignins were characterized and their results compared with those of the raw kraft lignin before modification. Molecular weight was determined using SEC-MALS, and the concentration of phenolic groups and antioxidant activity were measured. Structural changes were also monitored using FTIR. The results indicate that the enzymatic modification of lignin using laccase led to an increase in the molecular weight of the modified lignins, along with a decrease in the content of phenolic groups and a reduction in antioxidant activity. Based on the obtained results, it can be concluded that the action of the laccase enzyme resulted in lignin polymerization and the formation of new chemical bonds, leading to further aggregation.
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Production of extracellular polysaccharides by extremophilic prokaryotes
Bystrická, Klaudia ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
Predložená diplomová práca sa zaoberá štúdiom biotechnologickej produkcie extracelulárnych polysacharidov (EPS) vybraných termofilných a halofilných mikroorganizmov. Po primárnom screeningu vybraných kultúr bola experimentálna časť tejto práce ďalej venovaná halofilnému archea Haloferax mediterranei. Cieľom tejto práce bola produkcia a charakterizácia polymérov a posúdenie biotechnologického potenciálu použitých extremofilných kultúr ako producentov EPS. Počas kultivácie H. mediterranei boli použité 3 základné média, ktoré boli následne optimalizované v prospech produkcie EPS. Získané precipitáty boli podrobené analýze pomocou GPC, FTIR a UHPLC-UV-ESI-MS/MS, na základe čoho bolo možné bližšie charakterizovať získaný produkt. Tieto experimenty dokázali, že so stúpajúcou koncentráciou NaCl v médiu, klesá výťažok polysacharidov a ich produkciu nepodporujú ani vysoké koncentrácie MgSO4 a KCl v médiu. Hlavnou komponentou polysacharidov vyprodukovaných H. mediterranei, je manóza, no v závislosti od použitého média môže obsahovať aj glukózu, galaktózu, ribózu alebo N-acetylglukosamin.
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Study on biodegradation of synthetic biobased polyesters
Guziurová, Pavlína ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
Tato práce se zabývá degradačními vlastnostmi polyesterových polymerů (PBS s inkorporovaným dilinoleovým sukcinátem v různých hmotnostních procentech) v simulovaných tělních tekutinách a odhaluje menší změny vlastností. Byla zkoumána také bakteriální degradace a analýza ukazuje významnější rozdíly ve výsledcích gelové permeační chromatografie (GPC) a skenovacího elektronového mikroskopu (SEM). Zatímco převažuje abiotická degradace, bakteriální prostředí vede k vyšším úrovním degradace než simulované tělní tekutiny, což naznačuje potenciální využití v obalové technice nebo zemědělství. Nižší náchylnost na abiotickou degradaci však otevírá nové možnosti pro další výzkum mechanismů bakteriální a enzymatické degradace pro ekologicky udržitelné alternativy v různých průmyslových odvětvích.
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