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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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Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
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Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
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Biotechnological production of selected biopolymers employing Haloferax mediterranei
Strečanská, Paulína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is to study of production of selected biopolymers, polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by Haloferax mediterranei. The first part contains theoretical background including properties of biopolymers, advantages and purposes of commercial production, characteristics and positive aspects of cultivation of extreme halophile archaea Haloferax mediterranei. The experimental part is focused on study of effects of concentration of waste substrates, such as proteolytic whey hydrolysate and feather hydrolysate on PHA and EPS production. The effect of medium volume and its correlation with acceess of oxygen on biopolymer production were studied as well. PHA, in particul copolymer P(3HB-co/3HV) was analysed by gas chromatography with FID detection. The content of 3HV in PHA to 15,09 % was achieved using medium which contains 25 % proteolytic whey hydrolysate without glucose supplementation. Medium with 10 % of feather hydrolysate and 10 % of feather hydrolysate with 30 g/l glucose proved to be suitable in volume 50 ml in 250 ml Erlenmeyer flask. Haloferax mediterranei was able to produce biomass and accumulate PHA using protein substrate without need of saccharide supplementation.
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Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
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Biotechnological production of selected biopolymers employing Haloferax mediterranei
Strečanská, Paulína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is to study of production of selected biopolymers, polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by Haloferax mediterranei. The first part contains theoretical background including properties of biopolymers, advantages and purposes of commercial production, characteristics and positive aspects of cultivation of extreme halophile archaea Haloferax mediterranei. The experimental part is focused on study of effects of concentration of waste substrates, such as proteolytic whey hydrolysate and feather hydrolysate on PHA and EPS production. The effect of medium volume and its correlation with acceess of oxygen on biopolymer production were studied as well. PHA, in particul copolymer P(3HB-co/3HV) was analysed by gas chromatography with FID detection. The content of 3HV in PHA to 15,09 % was achieved using medium which contains 25 % proteolytic whey hydrolysate without glucose supplementation. Medium with 10 % of feather hydrolysate and 10 % of feather hydrolysate with 30 g/l glucose proved to be suitable in volume 50 ml in 250 ml Erlenmeyer flask. Haloferax mediterranei was able to produce biomass and accumulate PHA using protein substrate without need of saccharide supplementation.
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Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
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