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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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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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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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Biotechnological production of PHA by selected thermophilic bacteria
Marková, Lucie ; Slaninová, Eva (referee) ; Pernicová, Iva (advisor)
This thesis is mainly focused on the production of PHA copolymers and study of metabolism of the thermophilic genus Aneurinibacillus. Theoretical part mainly deals with general characterization and production of PHA and with PHA producing bacteria, especially the extremophiles. The last chapter talks about the bacterial genus Aneurinibacillus and previous knowledge about this PHA producer. The experimental part is focused on testing the PHA synthase from Aneurinibacillus, which can synthesize large scale of different monomers. The two bacterial isolates, AFN2 and AH30, were used to produce copolymers with unsusual monomers in their structure, such as 3HB, 3HV, 4HB, 4HV, 5HV and even 3HHx and 4HHx. This means that the PHA synthase has low substrate specifity. The production of copolymers was supported by adding precursors which encompass lactones, diols and organic acides. There were used two different types of medium. The mineral medium, which generate the environment with limited content of nitrogen, and complex medium, which provide to bacteria all required elements. It was observed that the genus Aneurinibacillus don´t require the nitrogen limitation to produce PHA. One of the most successful experiments was the cultivation of AFN2 on complex medium with added glucose and -valerolactone as precursor. The concentration of biomass was 4,2 g/l and the syntesized PHA reached 2,6 g/l. This copolymer was composed nearly of 60 mol. % monomer 4HB. In the last section of experimental part the two pair lacton-diol were selected to produce more PHA copolymer wich was later isolated and characterized. When the lactone was used as precursor, the resulting PHA copolymer reported higher molecular weight. This can be explained by alcoholytical activity of PHA synthase from bacterial genus Aneurinibacillus.
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