|
|
Identification and isolation of PHA producing bacteria
Pernicová, Iva ; Ondrejovič, Miroslav (referee) ; Rychtera, Mojmír (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.
|
|
|
Study on biotechnological potential of Pseudomonas thermotolerans
Vašířová, Kristýna ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor‘s thesis was to evaluate biotechnological potential of the bacterium Pseudomonas thermotolerans. The ability of bacterium to produce polyhydroxyalkanoates (PHA) and biosurfaktants was examined. Theoretical part deals with characteristic of extremophilic bacteria, PHA and biosurfactants. In the experimental part, the bacterium was tested for the presence of gene that catalyzes the synthesis of mcl-PHA (phaC1), using molecular technique PCR. Presence of the gene was confirmed by the experiment. Based on the result, PHA production conditions were optimized, such as selection of ideal media for production, suitable cultivation temperature and precursors. However, non of the tests confirmed the ability of production. Another of the experiments was to examine the bacterium’s ability to produce biosurfactants. Testing was realized in a suitable mineral medium with different carbon sources. Demonstrable production of biosurfactants was only on mineral medium supplemented by yeast extract and fructose. The thin layer chromatography (TLC) demonstrated, that the bakterium produces a mixture of monorhamnolipids and dirhamnolipids. However, higher portion is represented by dirhamnolipids.
|
|
|
Study on PHA production in extremophiles from genus Bacillus and related genuses
Reinohová, Nikola ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This Bachelor thesis is focused on study of production od polyhydroxyalcanoteas by extremophilic bacteria of genus Bacillus and related genera. In this thesis were studied microorganisms from german and czech collections Ureibacillus composti DSM 171951, Alkalibacillus haloalkaliphilus DSM 5271, Alicyclobacillus acidocaldarius DSM 446, Halobacillus halophilus CCM 3527, Thermobacillus composti DSM 18247, Bacillus licheniformis CCM 2206 and isolated microorganisms from natural sources Aneurinibacillus thermoaerophilus LK7, Aneurinibacillus thermoaerophilus F109, Aneurinibacillus thermoaerophilus AFn2, Geobacillus thermodenitrificans F101, Geobacillus thermodenitrificans F102, Geobacillus stearothermophilus A12, Geobacillus sp. AH11. The theoretical part describes extremophilic microorganisms, polyhydroxyalkanoates and their applications. In the experimental part, the detection of PHA production at the genotype level was performed using the PCR method, where the presence of first and fourth class PHA synthases was detected. Detection of presence of the 16SrRNA gene was performed by PCR. PHA production was also tested at the phenotype level, where the ability of utilization of different carbon sources and the ability of microorganisms to form 4HB and 3HV copolymers with different precursors at different temperatures was monitored. Copolymers are very interesting because of their properties, which make them suitable for a wide range of applications. The microorganism Aneurinibacillus thermoaerophilus AFn2 proved to be the best representative for PHA production in this work, producing PHA up to 1,99 g/l and 3HV copolymer up to 0,49 g/l.
|
|
|
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.
|
|
|
Identification of microbial pigments in evaporites using Raman spectroscopy: implications for astrobiology
Vítek, Petr ; Jehlička, Jan (advisor) ; Marshall, Craig P. (referee) ; Vandenabeele, Peter (referee)
Raman spectroscopy is a powerful tool for identification both inorganic and organic compounds including microbial biomolecules. Together with the fact, that it is considered to be the important nondestructive instrument for use on Mars within future robotic missions, it is necessary to assess its capabilities in scenarios relevant for both Martian and terrestrial conditions. In this work, the potential of Raman spectrometry was tested - including both bench-top laboratory systems as well as portable counterparts - to detect traces of life within evaporitic matrices through biomolecular identification. Due to their chemical and physical nature resulting in optical properties, pigments are important organic compounds in Raman spectroscopic analysis using visible excitation. Hence in this work we have focused on the Raman spectroscopic identification of pigments as biomarkers with relevance for investigation of life in both extreme terrestrial and potentially extraterrestrial environments. Results of methodical work are presented in Appendices I to III, dealing particularly with β-carotene as a model carotenoid pigment. The concentration limits of this biomarker in three different evaporitic matrices (halite, gypsum and epsomite) have been determined for artificially prepared powdered mixtures alone...
|
|
|
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.
|
|
|
Study on PHA production in extremophiles from genus Bacillus and related genuses
Reinohová, Nikola ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This Bachelor thesis is focused on study of production od polyhydroxyalcanoteas by extremophilic bacteria of genus Bacillus and related genera. In this thesis were studied microorganisms from german and czech collections Ureibacillus composti DSM 171951, Alkalibacillus haloalkaliphilus DSM 5271, Alicyclobacillus acidocaldarius DSM 446, Halobacillus halophilus CCM 3527, Thermobacillus composti DSM 18247, Bacillus licheniformis CCM 2206 and isolated microorganisms from natural sources Aneurinibacillus thermoaerophilus LK7, Aneurinibacillus thermoaerophilus F109, Aneurinibacillus thermoaerophilus AFn2, Geobacillus thermodenitrificans F101, Geobacillus thermodenitrificans F102, Geobacillus stearothermophilus A12, Geobacillus sp. AH11. The theoretical part describes extremophilic microorganisms, polyhydroxyalkanoates and their applications. In the experimental part, the detection of PHA production at the genotype level was performed using the PCR method, where the presence of first and fourth class PHA synthases was detected. Detection of presence of the 16SrRNA gene was performed by PCR. PHA production was also tested at the phenotype level, where the ability of utilization of different carbon sources and the ability of microorganisms to form 4HB and 3HV copolymers with different precursors at different temperatures was monitored. Copolymers are very interesting because of their properties, which make them suitable for a wide range of applications. The microorganism Aneurinibacillus thermoaerophilus AFn2 proved to be the best representative for PHA production in this work, producing PHA up to 1,99 g/l and 3HV copolymer up to 0,49 g/l.
|
|
|
Identification and isolation of PHA producing bacteria
Pernicová, Iva ; Ondrejovič, Miroslav (referee) ; Rychtera, Mojmír (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.
|
|
|
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.
|
|
|
Study on biotechnological potential of Pseudomonas thermotolerans
Vašířová, Kristýna ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor‘s thesis was to evaluate biotechnological potential of the bacterium Pseudomonas thermotolerans. The ability of bacterium to produce polyhydroxyalkanoates (PHA) and biosurfaktants was examined. Theoretical part deals with characteristic of extremophilic bacteria, PHA and biosurfactants. In the experimental part, the bacterium was tested for the presence of gene that catalyzes the synthesis of mcl-PHA (phaC1), using molecular technique PCR. Presence of the gene was confirmed by the experiment. Based on the result, PHA production conditions were optimized, such as selection of ideal media for production, suitable cultivation temperature and precursors. However, non of the tests confirmed the ability of production. Another of the experiments was to examine the bacterium’s ability to produce biosurfactants. Testing was realized in a suitable mineral medium with different carbon sources. Demonstrable production of biosurfactants was only on mineral medium supplemented by yeast extract and fructose. The thin layer chromatography (TLC) demonstrated, that the bakterium produces a mixture of monorhamnolipids and dirhamnolipids. However, higher portion is represented by dirhamnolipids.
|
guest ::
