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Study on metabolic properties of the bacterium Schlegelella thermodepolymerans with respect to biosynthesis of polyhydroxyalkanoates
Chvátalová, Viktorie ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This bachelor's thesis deals with the metabolic properties of the thermophilic bacterium Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates (PHA). The aim of the study was to understand the metabolism and utilization of different carbohydrates (xylose, cellobiose, glucose and their combinations) depending on the cultivation time. In the experimental part, two bacterial strains (M15344 and DSM15344) were used. The increase in optical density was monitored during the measurements for the bacteria. Furthermore, the content of P(3HB) from the biomass was determined using GC-FID and the molecular weight of the polymer was determined using SEC- MALS analysis. By measuring residual sugar using HPLC-RI, the course of carbohydrate utilization was determined as a function of time. The results of both bacteria were compared and evaluated. DSM15344 showed better results in P(3HB) production than M15344, and cellobiose and xylose appeared to be the most suitable substrates for P(3HB) production.
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Production of polyhydroxyalkanoates by bacterium Schlegelella thermodepolymerans
Černý, Vojtěch ; Černayová, Diana (referee) ; Pernicová, Iva (advisor)
The aim of this work was to study capability of various strains of thermophilic bacteria Schlegelella thermodepolymerans incorporate diverse monomer units to structure of polyhydroxyalkanoates (PHA). Precursors for production mcl-PHA, 4-hydroxybutyrate and 3-hydroxyvalerate were tested. The theoretical part deals with characteristic of studied polymers, properties and applications and usage of thermophilic bacteria in industry. In experimental part there were undergone cultivations with different precursors, which were added in different times and concentrations. Four bacterial strains S. thermodepolymerans from various public collections of microorganisms were used during these cultivations. Levulinic acid, added at start of cultivation, was chosen as the best precursor. Afterwards there was cultivation with concentration line to find out, whether concentration influence composition of copolymer. Increasing the amount of precursor led to decreasing quantity of biomass and PHA, but there were very interesting materials with high amount of 3HV. Addition 8 g/l of precursor led up to 52 %. Subsequently there was found out molecular height of polymers by size-excluion chromatography (SEC) combined with multi-angle light scattering (MALS).
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Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) employing selected members of the genus Aeromonas
Čeparová, Klára ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoates (PHA) production by Aeromonas spp. Four bacterial strains were selected for this study: Aeromonas aquatica (DSM 100827), Aeromonas bivalvium (DSM 19111), Aeromonas hydrophila (CCM 7770) and Aeromonas cavernicola (CCM 7641). The presence of phaC gene responsible for PHA synthesis was verified by polymerase chain reaction (PCR). In three of four selected strains (A. aquatica, A. bivalvium, A. hydrophila) the presence of phaC gene was confirmed. Strains A. cavernicola did not contain the phaC gene. PHA synthesis on different carbon substrates was further tested on strains with confirmed presence of the phaC gene. The aim of the thesis was to optimize the cultivation conditions of bacterial cultures for the PHA synthesis. The production of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) was monitored. The evaluation of polymer presence and concentration in biomass was determined by gas chromatography. For cultivation, decanoic acid, caproic acid, octanoid acid, glukose, sucrose, fructose were used as carbonaceous substrates. The highest of both PHB and the copolymer P(3HB-co-3HHx) were recorded by A. aquatica with addition of decanoid acid at a concentration of 1 g/l to the production medium. Among the sugar substrates, cultivation on sucrose was the most successful with, resulting in the highest biomass concentration.
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Evolutionary and genetic engineering of bacterial producers of polyhydroxyalkanoates
Nováčková, Ivana ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
This doctoral thesis deals with the topic of evolutionary and genetic engineering of polyhydroxyalkanoates (PHA) producing bacteria. Apart from these topics, the issue of biotechnological production of PHA on model hydrolysates of lignocellulosic biomass with the use of extremophilic microorganisms is also studied, as well as the development of an alternative method of PHA isolation. The themes were freely linked to previous experiments and reflected the currently solved projects in a working group. Doctoral thesis is prepared in the form of a commented discussion of published works, which are part of it in the form of appendices. Evolutionary engineering was mainly applied to the model PHA producing bacterial strain Cupriavidus necator H16. By adaptation to levulinic acid, isolates producing copolymer P(3HB-co-3HV) with a higher content of the 3HV fraction were obtained, which leads to improved properties of the polymer for further processing. As well as culture growth also the amount of total PHA in the biomass was higher. By long-term adaptation of the same strain to osmotic stress and the presence of copper ions, the isolates which are characterized in the second publication, were obtained. Based on obtained data, it was possible to observe differences in the adaptation process, where the adaptation to osmotic stress was gradual, while a significant step in the increase of biomass and PHA signaling faster adaptation was observed for copper. Based on the analyses, the significant role of PHA in the adaptation of the C. necator H16 strain to the tested stressors was discussed, it did not consist only in the increase in the amount of polymer in the biomass, but also in enhancement of whole PHA cycle, which also leads to an increase of the pool of monomeric units showing protective functions. By adaptation to -captolactone, a unique precursor of 4HB, the copolymer P(3HB-co-4HB) was obtained. The properties of this copolymer are again more favorable than of the homopolymer P(3HB), even with a low content of 4HB, which we also achieved in a laboratory bioreactor. A further increase in the 4HB fraction could be achieved using deletion mutants with the absence of relevant genes, which is discussed more in the text. The production of PHA on models of lignocellulosic biomass hydrolysates originating from, for example, the food industry was tested in combination with the use of extremophile producers, when the preference of the contained monosaccharides (hexoses, pentoses) for individual producers was discussed. For the purpose to get closer to real hydrolysates, the resistance of the strains to relevant potential microbial inhibitors was also tested. The susceptibility of halophilic and thermophilic PHA producers to osmotic stress was used in the development of an alternative isolation approach that would reduce the economic and ecological burden of the process compared to standard extraction using chlorinated solvents. Application of SDS detergent at low concentrations while simultaneously exposing the cells to higher temperatures led to the gain of high purity polymer without loss of yield. The recycling process of used SDS is also a possibility.
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Study on metabolism of polyhydroxyalkanoates in Rhodospirillum rubrum
Rubanová, Blanka ; Vodička, Juraj (referee) ; Slaninová, Eva (advisor)
This bachelor thesis deals with the study of polyhydroxyalkanoate (PHA) metabolism in Rhodospirillum rubrum under different cultivation conditions. The conditions differed in the aeration rate (aerobic, anaerobic, microaerobic) and in the presence of a light source (light cultivation, dark cultivation). First, different volumes of medium were suggested and tested for the fastest and the most suitable inoculum cultivation. Subsequently, cultures of Rhodospirillum rubrum under different conditions in terms of PHA content were compared by gas chromatography. In addition to gas chromatography, other complementary methods were used for better characterization of bacterial growth and metabolism, such as flow cytometry, UV-VIS spectroscopy (transmission and diffusion transmission modes). Fluorescence microscopy and cryo-scanning electron microscopy were used in terms of cell morphology. Furthermore, the ability of bacteria to synthesize color pigments (carotenoids and bacteriochlorophyll a) was investigated. The bacteria were cultivated on different types of media (liquid, solid), the color pigments were subsequently extracted, and their concentrations were calculated. UV-VIS spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) were also used to measure color pigments.
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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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Behaviour of microorganisms using microcalorimetry
Piletskaya, Maryia ; Sedláček, Petr (referee) ; Krouská, Jitka (advisor)
The diploma thesis deals with the study of the behavior of a microorganism with the help of microcalorimetry. The theoretical part of the thesis deals mainly with the characterization of the microorganism Rhodospirillum rubrum selected for experimental work and the application of microcalorimetry in microbiology. The second goal was to screen the growth of the bacterial strain R. rubrum using different substrates, and subsequently, based on experimental results, to select the most suitable substrate, and optimize its concentration and aeration of the medium for the microorganism using microcalorimetry. Hexanoate, malate, acetate and fructose were used to examine the most suitable substrate by microcalorimetry. Another goal was data processing and discussion of the advantages and disadvantages of substrate selection and media aeration with respect to bacterial strain growth. Hexanoate was found to be the best substrate for growth, followed by optimization of the concentration and aeration of the medium. The results of the microcalorimetric measurement were confirmed by spectrophotometric study of the growth curve of the investigated bacteria. The effect of osmotic pressure on bacteria and the dependence of their survival on the amount of polyhydroxyalkanoates (PHA) in the cell were also studied. The amount of PHA in the biomass was determined chromatographically.
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Biotechnological production of PHA copolymers containing 4-hydroxybutyrate
Kovářová, Radka ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.
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Evolutionary engineering of cyanobacteria with respect to PHA accumulation
Vašířová, Kristýna ; Slaninová, Eva (referee) ; Samek, Ota (advisor)
The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.
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Study on PHA accumulation by cyanobacteria
Pacasová, Viktorie-Alexandra ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis was focused on studying the influence of selected stress factors on PHB production in strains of cyanobacteria Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Cyanobacterial stains were cultivated in multicultivator and as a stress factor an increased salt (sodium chloride) as well as a mixture of acetate and -butyrolactone was selected. The PHB content in cyanobacteria was determined by gas chromatography. In addition, the content and presence of pigments in cyanobacteria strains was determined by pigment extraction as well as using direct spectroscopy of whole cells. When cultivated with an increased salinity, the hypothesis of an increased PHB production was not confirmed most likely due to and overstressing the Synechocystis salina CCALA 192 cultures with increased salt content, constant illumination of multicultivator and by nitrogen limitation due to the used medium. When cultivated with the mixture of acetate and -butyrolactone the stains were analysed in the same way as in the cultivation with an increased salinity. In this case, the presence of 3-hydroxybutyrate and 4-hydroxybutyrate copolymer was determined in the stain cultivated with the mixture of acetate and -butyrolactone. During this cultivation, a positive effect of initial concentration of cells in the cyanobacterial cultures combined with continuous illumination and the type of nitrogen-limiting type of medium on the growth rate of selected stains was observed.
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