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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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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 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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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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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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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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Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
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Biodegradability of bacterial natural and modified polyesters and their composites
Pala, Martin ; Babák, Libor (referee) ; Obruča, Stanislav (advisor)
Presented work was focused on biodegradability of bacterial natural and modified polyesters and their composites. The first part of the work was focused on study of influence of PHA granules structure on their biodegradability using selected enzymes and influence of physiological conditions on PHA stability. Overall, tested polymer either in crytalinne or amorphous phase seems to be rezistent to attack of seleced hydrolytic enzymes such as lipases or proteases and is stable in simulated physiological fluids as well. Because of thies results, it is possible to use tested PHA materials in biomedical applications requiring rather resistant biomaterials. Second part of the work was focused on microbial degradation of modified PHA materials considering their potential environmental impact. Both mixed thermophillic culture originaly used in wastewater treatment plant and bacterial strain Delftia acidovorans were employed for biodegradation tests. Composites containing chlorine PHB and PHB films modified using plasticizers were tested. Films containing chlorine PHB cause inhibition of biomass growth to both tested cultures. The highest rate of degradation (31%) was observed in presence of bacterial culture with film containing 10% chlorine PHB. The results show that used microbial population is important factor affecting biodegradability.
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Employment of bacterium Tepidimonas taiwanensis for production of polyhydroxyalkanoates on waste substrates
Vidláková, Michaela ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on screening PHA production using thermophilic bacterium Tepidimonas taiwanensis and on the study of possible use of grape pomace, molasses and waste paper as a cheap carbon substrate for culturing the characterized bacteria. At first, testing of basic cultivation parameters was performed, including carbon substrate concentration, oxygen availability, ability to utilize nitrogen sources and selected disaccharides. PHA production from waste substrates was tested in three ways. In the first, pre-prepared solids-free hydrolysates from raw materials were used as the carbon source. The second and third procedures were performed by dosing waste materials directly into the mineral media, which differed only in the presence or absence of the enzyme preparation enabling release of fermentable sugars. The most intensive increase in culture and the highest production of PHA was recorded on grape hydrolyzate. The biomass concentration in this sample reached up to 4.8 g/L with a content of 59 % PHA. On the other hand, the addition of grape marc directly to the production medium did not work at all, which was probably due to the presence of a large amount of inhibitory substances from the pomace. The situation was similar with molasses and waste paper, where the bacterium Tepidimonas taiwanensis was able to grow and possibly produce PHA only to a small extent. The work also managed to characterize the effect of temperature and pH on the activity of the enzyme cocktail Viscozyme L and to determine the temperature and pH optimum PHA synthase of the bacterial strain Tepidimonas taiwanensis in the cell lysate.
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Stress resistance of PHA accumulating bacteria against conditions associated with gastrointestinal tract
Kovářová, Radka ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.
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