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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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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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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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Metabolism of Bacterial Cells and the Effect of Stress on Biosynthesis of PHA
Kučera, Dan ; Kráčmar, Stanislav (referee) ; Ondrejovič,, Miroslav (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of polyhydroxyalkanoate biosynthesis as a microbial product with the potential to replace current conventional plastics made from petroleum. The dissertation thesis is elaborated in the form of a discussed set of already published publications, which are then part of the thesis in the form of appendices. The work builds on relatively extensive knowledge in the field of polyhydroxyalkanoate production and brings new facts and possible strategies. Various possibilities of analysis of polyhydroxyalkanoates using modern methods were tested in this work, which brings especially speed, which can be crucial in real-time evaluation of production biotechnological process. Raman spectroscopy has proven to be a very promising technique for rapid quantification of PHA. Furthermore, the work deals with valorisation of waste of food and agricultural origin. Emphasis is placed on methods of detoxification of lignocellulose hydrolysates. In this context, adsorption of inhibitors to lignin was first used as an alternative to other detoxification techniques. Due to detoxification, selected production strains Burkholederia cepacia and B. sacchari were able to utilize softwood hydrolyzate for PHA production. In the next part of the work was also tested the possibility of using chicken feathers as a complex source of nitrogen. Evolutionary engineering was also used as a possible strategy to eliminate the inhibitory effect of levulic acid as a microbial inhibitor that results from the hydrolysis of lignocellulosic materials. Adaptation experiments were used to develop strains exhibiting higher resistance to levulic acid and the ability to accumulate a higher 3HV copolymer from the original wild-type C. necator strain. Another promising approach tested in the work was the use of extremophilic microbial strain, which leads to a reduction in the cost of biotechnological production. Selected Halomonas species have shown high potential as halophilic PHA producers. The final part of the thesis was devoted to the selection of the production strain with regard to the properties of the resulting PHA. The Cupriavidus malaysiensis strain was selected to produce a P(3HB-co-3HV-co-4HB) terpolymer which revealed significant differences in material properties over P3HB.
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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 selected thermophilic bacteria
Reinohová, Nikola ; Sedláček, Petr (referee) ; Pernicová, Iva (advisor)
This thesis is focused on adaptive evolutionary engineering in the thermophilic bacterium Schlegelella thermodepolymerans M15344. The theoretical part deals with extremophilic microorganisms, biotechnological applications as well as polyhydroxyalkanoates and evolutionary engineering. In this work, adaptation of this microorganism to 40 °C and 60 °C was carried out using glucose and xylose substrates. Furthermore, repeated-batch cultivation at 50 °C on glucose and xylose was performed. In these experiments, the concentration of PHA produced in the biomass was determined by gas chromatography, and the concentration of residual sugars in the medium was determined by a method using DNS. Production of polyhydroxyalkanoates in the adapted strains from each experiment was compared with the non-adapted strain. Subsequently, the PHA produced by individual bacteria was studied using the SEC-MALS method, where the effect of adaptation on molecular weight was observed. The highest producing bacteria was adapted to 40 °C and the substrate was glucose. In this case, the biomass value was 6,74 g/l with a PHA percentage of 56.5 wt%. The bacteria was also adapted to 60 °C where it was able to grow and produce unlike the original bacteria.
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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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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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Metabolism of Bacterial Cells and the Effect of Stress on Biosynthesis of PHA
Kučera, Dan ; Kráčmar, Stanislav (referee) ; Ondrejovič,, Miroslav (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of polyhydroxyalkanoate biosynthesis as a microbial product with the potential to replace current conventional plastics made from petroleum. The dissertation thesis is elaborated in the form of a discussed set of already published publications, which are then part of the thesis in the form of appendices. The work builds on relatively extensive knowledge in the field of polyhydroxyalkanoate production and brings new facts and possible strategies. Various possibilities of analysis of polyhydroxyalkanoates using modern methods were tested in this work, which brings especially speed, which can be crucial in real-time evaluation of production biotechnological process. Raman spectroscopy has proven to be a very promising technique for rapid quantification of PHA. Furthermore, the work deals with valorisation of waste of food and agricultural origin. Emphasis is placed on methods of detoxification of lignocellulose hydrolysates. In this context, adsorption of inhibitors to lignin was first used as an alternative to other detoxification techniques. Due to detoxification, selected production strains Burkholederia cepacia and B. sacchari were able to utilize softwood hydrolyzate for PHA production. In the next part of the work was also tested the possibility of using chicken feathers as a complex source of nitrogen. Evolutionary engineering was also used as a possible strategy to eliminate the inhibitory effect of levulic acid as a microbial inhibitor that results from the hydrolysis of lignocellulosic materials. Adaptation experiments were used to develop strains exhibiting higher resistance to levulic acid and the ability to accumulate a higher 3HV copolymer from the original wild-type C. necator strain. Another promising approach tested in the work was the use of extremophilic microbial strain, which leads to a reduction in the cost of biotechnological production. Selected Halomonas species have shown high potential as halophilic PHA producers. The final part of the thesis was devoted to the selection of the production strain with regard to the properties of the resulting PHA. The Cupriavidus malaysiensis strain was selected to produce a P(3HB-co-3HV-co-4HB) terpolymer which revealed significant differences in material properties over P3HB.
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Evolutionary engineering of polyhydroxyalkanoates producing bacteria
Nováčková, Ivana ; Kovalčík, Adriána (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.
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