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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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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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Use of molecular techniques for study of thermophilles
Dvořáková, Dominika ; Kouřilová, Xenie (referee) ; Buchtíková, Iva (advisor)
Polyhydroxyalkanoates are microbial storage polymers that represent a green alternative to petrochemical plastics. However, their high production cost limits wider industrial utilization. Use of waste materials as a substrate and/or utilization of extremophilic organisms is an option to reduce production costs. The strain of thermophilic bacteria Aneurinibacillus appears to be a promising producer because of its ability to synthesize wide range of non traditional copolymers. In addition, PHA synthesis is independent of nutrient limitation, which was the subject of this study. Experiments were focused on the expression of genes involved in PHA synthesis in the bacterium Aneurinibacillus sp. AFn2 on different types of production media. The presence of selected genes was verified using classical PCR. After that the cultivation experiments were performed on mineral and complex media to simulate environments with different nutrient availability. Biomass concentration was determined gravimetrically from the samples collected in time during the growth curve measurement. PHA content was measured simultaneously using gas chromatography. Finally, the expression of the studied genes was analysed by RTqPCR. Significant differences were observed in the transcription of individual genes depending on the media type and time. The results confirmed the ability of Aneurinibacillussp.AFn2 to produce PHA independently on nutrient limitation, however, the production rate and cell filling depended on the media type. This fact may be related to the different course of expressed genes involved in PHA synthesis. A higher percentual yield of P(3HB) in cells were observed in complex media along with an increasing trend in the overall gene expression. On the other hand, in mineral medium, the transcription of individual genes only decreased after initial strong expression, and the cell filling of P(3HB) was also lower. The findings lead to understanding not only the expression of selected genes but also the overall synthesis of PHA. At the same time, it offers the way to optimize production with the aim of reducing cost and expanding their market applications.
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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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