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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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Characterization of bacterial strains obtained in evolutionary engineering
Hrabalová, Vendula ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with application of evolutionary engineering on PHA producing bacterial strains. Two bacterial strains, Cupriavidus necator H16 and Halomonas halophila, were chosen for the evolutionary experiments. Copper cations (Cu2+) and sodium chloride (NaCl) were chosen as the selective pressure for C. necator H16; acetic acid (AA) and levulinic acid (LA) for Halomonas halophila. The adapted strains were during long-time evolutionary experiments characterized by GC-FID and SEC-MALS. The growth of the adapted strains was studied by the mean of optical density measurement. The amount of viable cells was determined by spectral FC after their expositon to selected stress factors. Specific enzyme activities of enzymes involved in citrate and glyoxalate cycle, enzymes generating NADPH, LA metabolism enzyme and PHA biosynthesis enzymes were determined. The adapted strains were compared with the wild-type of strains. The successfull adaptation of C. necator H16 adapted to Cu2+ was detected. Biomass and PHA production of both wild and adapted H. halophila strains cultivated in lignocellulosis waste were determined. It was found out that H. halophila adapted to the LA is capable of producing more PHA than the wild strain of this bacteria.
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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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Acetic acid as a side product of metabolism of PHA producing bacteria
Ryšavá, Lenka ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
The Bachelor´s thesis deals with experimental study production of acetic acid as a side product of metabolism of PHA producing bacteria. Experimental part of work deals with the impact of used substrate on the biomass growth, PHA production and the formation of organic acids in strains that can potentially find their application in industrial production of PHA – Cupriavidus necator H16, Burkholderia sacchari, Burkholderia cepacia. The highest yields of organic acid have been reached on Burkholderia sacchari when cultivated on mineral medium with glucose. The last part of the thesis is focused on determining the viability of micro organisms using flow cytometry where bacteria Cupriavidus necator H16, Burkholderia sacchari, Burkholderia cepacia were exposed to selected stress factors.
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DNA Isolation and Analysis Focused on Microorganisms Important in Food Production
Čutová, Michaela ; Obruča, Stanislav (referee) ; Fojtová,, Miloslava (referee) ; Brázda, Václav (advisor)
Identification of bacterial DNA consists from several steps: cell lysis, isolation and purification of DNA, precipitation by ethanol, identification of bacterial strain by PCR or other molecular biology methods. Each step must be optimised. Nucleic acids can be isolated from cells using magnetic particles. The molecules of DNA are bound to the surface of magnetic carriers by electrostatic interaction, and then they are eluted into buffer. The aim of the work will be to optimize individual steps of identification of bacterial DNA: cell lysis, DNA isolation, characterization of solid magnetic carriers functionalized by amino groups for nucleic acids isolation. The presence of DNA will be verified using agarose gel electrophoresis and the amount of eluted DNA will be determined spectrophotometrically. The quality of isolated DNA will be proved by their amplification using polymerase chain reaction (PCR). Furthermore, the thesis focuses on the study of secondary structures of nucleic acids – cruciforms structures and quadruplexes. These structures are involved in the regulation of cellular processes and their appearance is associated with cancer development and neurodegenerative diseases. In silico genome analysis was performed on important food industry microorganisms. The microorganisms genomic sequences were obtained from the NCBI (National Center for Biotechnology) database. The Palindrome Analyzer and G4 Hunter software were used for the analysis.
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Involvement of polyhydroxyalkanoates in stress response of bacteria during late stationary phase
Šuráňová, Zuzana ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase. Bacteria Cupriavidus necator H16 (able to produce PHA) and bacteria Cupriavidus necator H16/PHB-4 (unable to produce PHA) were used for the experiment. In the theoretical part, the polyhydroxyalkanoates and a stress response of bacteria were reviewed. In the experimental part of the work, the involvement of polyhydroxyalkanoates into stress response of bacteria in the late stationary phase against selected stress factors was studied. A resistence against various stress conditions of bacteria was studied. During long term cultivations a culture viability as well as PHA distribution among bacterial populations were assessed by using flow cytometry and the PHA content in biomass was analyzed by gas chromatography with FID detector.. Based on the results obtained in this work, it was found that the PHA acumulating bacteria Cupriavidus necator H16 is capable to survive carbon substrate limitations better than the bacteria Cupriavidus necator H16/PHB. Further, Cupriavidus necator H16 also revealed higher resistence against various stress factors such as ethanol treatment and freezing.
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