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Biotechnological production of PHA by selected bacterial isolates
Matějka, Filip ; Šedrlová, Zuzana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the production of polyhydroxyalkanoates (PHA) using thermophilic bacterial isolates with designation 34, 35 and BŽ. Bacterial straines were isolated from activated sludge and compost The theoretical part contains a description of PHA, extremophilic bacteria and detection techniques for the determination of polyhydroxyalkanoates. In the experimental part, the presence of the phaC gene, which is crucial for the ability to produce PHA, was first determined by PCR and bacterial strains were also subjected to DNA sequencing of 16S rRNA gene which enabled preliminary taxonomical classification of the isolates. In the next part of the experimental work, the conditions for biomass growth and PHA production were optimized. Suitable carbon source, the ideal temperature for cultivation and the influence of precursors on the production of copolymers were studied and identified. The composition and proportion of PHA were determined spectrophotometrically and by GC-FID. Finally, visual screening of PHA accumulation inside bacterial cells was performed using fluorescence microscopy.
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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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Screening of biotechnological potential of selected members of the genus Geobacillus and other related genuses
Kouřilová, Xenie ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.
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Screening of extremozyme production of selected extremophilic PHA producers
Dyagilev, Dmitry ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
This bachelor thesis deals with the screening of the production of extracellular hydrolytic enzymes in thermophilic microorganisms of the genera Aneurinibacillus, Brevibacillus, Chelatococcus, Pseudomonas, Schlegelella, Tepidimonas and Caldimonas. The ability of selected enzymes, namely proteases, lipases, amylases, xylanases, cellulases and pectinases, was tested in the investigated microorganisms. Such testing made it possible to assess in which microorganisms the production of specific enzymes can be observed. Based on the results of the screening, it was found that Schlegelella aquatica LMG 23380, Tepidimonas fonticaldi LMG 26746 and the investigated microorganisms of the genus Chelatococcus did not show the ability to produce any of the tested enzymes extracellularly. In natural isolates of Brevibacillus borstelensis LK 99 and Aneurinibacillus thermoaerophilus LK 102, only the ability to produce lipolytic enzymes was detected. The isolate Brevibacillus borstelensis Bz acts as a universal producer of all selected extremozymes. Enzyme activity was determined for selected producers. The bacterium Brevibacillus borstelensis Bz proved the ability to produce all six selected hydrolytic enzymes and has the highest activity of lipases, xylanases, cellulases and pectinases from the tested microorganisms. The highest proteolytic activity was measured in Thermomonas hydrothermalis DSM 14834 when cultured on skimmed milk powder.
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Novel approaches for PHA isolation from bacterial biomass
Dlouhá, Karolína ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The aim of this diploma thesis was study of the isolation of polyhydroxyalkanoates using a commercial surfactant and selected biosurfactants, which were sophorolipids, coconut soap, lecithin, lauryl glucoside, coco glucoside and cocamidopropyl betaine. PHA was isolated from Schlegelella thermodepolymerans DSM 15344, where the amount of NaCl in the production medium was first optimized. The molecular weight of the isolated polymer was analyzed by SEC-MALS. The largest structural changes were recorded for coconut soap. Possible impurities in the isolated polymer were analyzed by infrared spectrometry (ATR-FTIR). The least protein contamination of the polymer was recorded with coconut soap. From the above biosurfactants, coconut soap, lauryl glucoside and coco glucoside were selected, because the highest purity of PHA was obtained. However, coconut soap had the greatest potential. The isolation temperature and surfactant concentration were optimized for selected biosurfactants. The best results were obtained at 90 °C and a concentration of 5 g/L. Furthermore, the versatility of the isolation method was tested using selected biosurfactants on various thermophilic microorganisms, which were Chelatococcus composti DSM 101465, Schlegelella thermodepolymerans DSM 15264, Tepidimonas taiwanensis LMG 22826 and Aneurinibacillus thermoaerophilus H1.
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Utilization of selected extremophilic bacteria for bioplastic production
Vidláková, Michaela ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on the study of the biotechnological potential of thermophilic bacteria Tepidiphilus thermophilus, Tepidimonas taiwanensis and Tepidimonas fonticaldi. The aim of this work was to investigate the ability of bacteria to produce polyhydroxyalkanoates (PHA). The theoretical part includes the characteristics of PHA and extremophilic bacteria. In the experimental part, the investigated bacteria were tested by molecular technique PCR for the presence of the phaC gene encoding for the first class PHA synthase. The presence of the phaC gene was confirmed in bacterial strains Tepidiphilus thermophilus and Tepidimonas taiwanensis. The ability of PHA production on various carbon sources was examined for all the selected microorganisms, optimal temperatures for cultivation were determined, and the ability to synthesize copolymers was investigated by adding suitable precursors to the production medium. The most intensive PHA production was recorded in the bacterial strain Tepidimonas taiwanensis, but other strains were also able to produce PHA. The ability to produce P(3HB-co-3HV) or P(3HB-co-4HB) copolymers has also been demonstrated in some strains. A significant amount of P(3HB-co-3HV) was obtained using the Tepidimonas taiwanensis strain. Morever, Tepidimonas taiwanensis strain was also tested for its ability to use various model lignocellulose hydrolysates to form PHA. The highest PHA gain was obtained using model hydrolysate of softwood. Furthermore, the ability of proteases production by selected bacterial strains was tested. The presence of these enzymes was detected in the Tepidimonas taiwanensis strain, which was followed by determination of the temperature optima of the proteases, the pH optima of the proteases and also of their molecular weights.
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Biotechnological production of PHA from waste substrates
Krejčiřík, Roman ; Hrabalová, Vendula (referee) ; Obruča, Stanislav (advisor)
The presented bachelor thesis deals with the study of polyhydroxyalkanoates using wine grape pomace extract. The thesis aims to determine whether it is possible to use wine grape pomace extract as a substrate for polyhydroxyalkanoates (PHA) production. For this research, four thermophilic bacterial strains were selected such as Schlegelella thermodepolymerans, Caldimonas meghalayensis, natural isolate labeled as 34, and Tepidimonas taiwanensis, from which the most suitable bacterium was subsequently selected. The best transformation of residual sugars showed T. taiwanensis, and therefore it was used for all the further fermentation. The extracts were prepared out of wine marc of Veltlínské červené rané, Müller Thurgau, Pálava, Sauvignon, Rulandské bílé, Frankovka (rosé), Frankovka (red wine). The highest PHA concentration was detected after fermentation with concentrated extracts of Sauvignon and Rulandské bílé. Fermentation processes in other extracts were most likely inhibited by the components they contained. Optimization was performed by dilution of extracts with water. The highest concentrations of PHA while using dilute extracts were achieved with the extract from different kinds of Frankovka (rosé) and Frankovka (red wine).
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Thermophilic Microorganisms Application for Lignocellulose Materials Biodegradation
Klašková, Lenka ; Zemanová, Jana (referee) ; Babák, Libor (advisor)
The plant cell wall consists of several layers: cellulose, hemicellulose, lignin and pectin. These biopolymers are degraded by many microorganisms. Extracellular enzymes are used for biodegradation by microorganisms. This thesis was focused on studying the impact of cultivation conditions on the production of extracellular enzymes at carboxymethyl cellulase and pectin when a mixed thermophilic culture containing Bacillus and Thermus microorganisms is used. The cultivation was carried out in flasks on a shaking machine with a shaking speed of 99 min-1 at a temperature of 60°C. The monitoring covered cellulolytic and polygalacturonase activities, protein concentration by the Biuret method, concentration of reducing substances by the Somogyi and Nelson methods, and the temperature optimum.
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Whey utilization with thermophilic microorganisms
Rychová, Alexandra ; Vítová, Eva (referee) ; Babák, Libor (advisor)
This diploma thesis studies the utilization of whey using thermophilic bacteria of the genus Thermus and Geobacillus. The whey stripped off proteins was used as a cultivation medium during experiments. The cultivation took place in the Erlenmeyer flasks, to assess the optimal conditions for microrganism’s growth. During the cultivation in the bioreactor, growth curves were established. The amount of whey utilization was assessed by analytical methods that determine the concentration of reducing saccharides (lactose) and chemical oxygen demand (COD) while studying the optimal conditions and a method determining the concentration of reducing saccharides during growth curves analysis.
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Involvement of polyhydroxyalkanoates in stress response of thermophilic bacterium Schlegelella thermodepolymerans
Berger, Matyáš ; Nováčková, Ivana (referee) ; Slaninová, Eva (advisor)
This bachelor's thesis is focused on investigating the effect of different stress factors on the bacteria Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates. In this case, in the form of poly(3-hydroxybutyrate). The selected stress factors were temperature shock, repeated freezing and defrosting. The last stress factor was osmotic stress, caused by sodium chloride solutions of different concentrations. In the theoretical part, polyhydroxyalkanoates, thermophilic organisms, mechanisms of individual stresses and methods of analysis of polyhydroxyalkanoates were characterized. In the experimental part, two strains of the thermophilic bacteria Schlegelella thermodepolymerans were compared, namely M 15344 and DSM 15344. The stress resistance of both strains was compared by measuring viability using flow cytometry. During the measurement, the increase in optical density was monitored, and the content of P(3HB) in the biomass was determined using GC-FID. Morphological changes caused by stresses were observed using Cryo-SEM and STEM electron microscopy methods. The DSM 15344 strain showed better growth and P(3HB) production than the M 15344 strain, which, on the other hand, showed better stress resistance.
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