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production of PHA by selected extremophilic bacteria
Šarköziová, Patrícia ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor´s thesis deals with production of polyhydroxyalkanoates (PHA) by thermophilic bacterial strains of Chelatococcus composti and Chelatococcus sambhunathii. We were finding out the best conditions for production of PHA granules inside the cells. In the beginning of the experiment we employed PCR to detect the phaC gene, encoding for PHA synthase, which is requisite for production of PHA. We also analyse the best source of carbon, optimal temperature for production of polyhydroxyalkanoates and influence of chosen precursors to grow of biomass and production of copolymers. The last part of this experimental thesis deals with research utilization of selected model hydrolysates. Glucose was the best source of carbon for both bacteria Chelatococcus composti and Chelatococcus sambhunathii and optimal temperature for grow and production of P(3HB) was 45°C. The applied precursors inhibited cell grow, but both bacterial strains Chelatococcus composti and Chelatococcus sambhunathii could produce copolymer P(3HB-co-3HV) after additiong of precursors sodium propionate and valeric acid. For both bacterial strains was the most useful model hydrolysates, which contains the biggest part of glucose. Morever, bacterial cells containing PHA granules were observed by fluorescence and transmission electron microscopy.
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Preparation of biodegradable films from PHA copolymers and evaluation of their application potential
Drotárová, Lenka ; Přikryl, Radek (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified.
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Composting of biodegradable polymers
Hollá, Tereza ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
This master’s thesis deals with the study of composting of polyhydroxybutyrate (PHB) and polylactic acid (PLA). The experimental part focuses on the composting test based on the IS/ISO 20200 standard, in which biopolymers were degraded within 8 weeks. The advantage of the composting test was the unique use of bags for polymer granules, which simplified the handling of samples. The placement of the polymers ensured an even load of compost, which simulated the landfill environment, which also made this experiment exceptional. The use of different methods of analysis ensured the investigation of the influence of compost on the degradation of polymers, but also the influence of polymers on the compost microbiome. In the compost environment all of the studied polymers degraded. After 8 weeks the weight loss of approximately 40 % was detected in PHB. Amorphous PLA degraded completely in the 5th week of experiment, while semicrystalline PLA lost approximately 70 % of its weight in 8 weeks. Using SEM images and SEC analysis, we conclude that PHB degradation was predominantly biological. Degradation of PLA samples was found to be predominantly abiotic. Analysis of compost samples revealed that the enzymatic activity of esterases increased in composts with polymers compared to compost without polymers. The effect of polymers on the microbiome in compost was analyzed using Biolog EcoPlates™. Compost with PHB showed a high ability to adapt to various substrates and microbiome expanded during composting. In the case of a sample with semicrystalline PLA, it was found that in the last week of composting, the ability of microorganisms to adapt and use substrates decreased, which indicates a possible negative effect of this polymer on compost development.
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Polyhydroxyalkanoates and their role in bacterial biofilms
Rucká, Markéta ; Samek,, Ota (referee) ; Obruča, Stanislav (advisor)
This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.
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Bioproduction, Characterization and Application of Selected Biopolymers
Benešová, Pavla ; Pekař, Miloslav (referee) ; Čertík, Milan (referee) ; Márová, Ivana (advisor)
Proposed doctoral thesis deals with microbial production of biopolymers (polysaccharides and polyesters) using renewableble inexpensive resources as carbon and nitrogen source. At first, production of extracellular metabolites, mainly pullulan and poly-L-malic acid, by polymorphic microorganism Aureobasisium pullulans was studied. Further part of thesis was focused on production of bacterial biopolyesters polyhydroxyalkanoates (PHA). PHA are produced in the form of intracellular granules by various bacteria species. During the study of PHA production, sythesis of poly(3-hydroxybutyrate) and technologically significant copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was investigated as well with regard to waste substrate utilization in biorafinery concept. Spent coffee ground seemed to be applicable waste in biorefinery proces. One of the coffee wastes used for PHA production was spent coffee ground hydrolysate which was converted to PHA by Burkholderia cepacia. Moreover, copolymer of 3HB and 3HV was accumulated when SCGH was used as a sole carbon source, no precursor had to be added. Another coffee waste, which was utillized by Cupriavidus necator H16, is represented by oil extracted from spent coffee grounds. Hydrolysis proces of waste substrates had to be optimized, to reach the highest possible saccharide concentration. Due to the hydrolysis process several toxic compounds can be formed, hence, influence of hydrolysis procesings on polyphenols content in hydrolysates was monitored. Waste substrates were also tested as potentional complex nitrogen source for PHA production. Hydrolysates of cheese whey and chicken feather were used as inexpensive complex nitrogen source for PHA production by C. necator, when waste frying oil was used as a carbon substrate. The finnal part of the thesis is dealing with possibilities how the PHA can be processed into atractive aplication forms. Liposomes with partial content of PHB had been prepared, moreover, for improving of antioxidant activity and antimicrobial effect the waste coffee oil was added. Micro-fibrous PHB materials were prepared for the purposes of water filtration. Higher filtration effectivity was observed when composite PHB fibrous material containing active charcoal or metal oxides was used. Important part of thesis is represented by study of degradation processes of prepared PHA-based aplication forms. Various microorganisms were tested on PHA depolymerases production. Biodegradability of PHA materials was tested in of composting conditions by standard composting test IS/ISO 20200. Changes in mechanical properties of polyurethan, where polyol was partialy replaced by PHB, were tested depending on the exposure of composting condition.
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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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Production of mcl-PHA by selected bacteria
Eremka, Libor ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates with medium chain length (mcl-PHA). For production two bacterial strains were used: Pseudomonas putida and Pseudomonas aeruginosa. Theoretical part of bachelor thesis deals with the biosynthesis of polyhydroxyalkanoates, their material properties and microbial producers. In experimental part selected bacteria were cultivated on different substrates to evaluate their ability to utilize various carbon sources and accumulate mcl-PHA. Content and composition of mcl-PHA accumulated in biomass were analyzed by gas chromatography with FID. Pseudomonas putida produced more mcl-PHA in medium containing canola oil as sole carbon source when compared with Pseudomonas aeruginosa. P. putida produced 2,11% mcl-PHA in medium containing glycerol as carbon source.
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Study on gene expression in Cupriavidus necator and other selected polyhydroxyalkanoates producers
Centnerová, Radmila ; Šedrlová, Zuzana (referee) ; Pernicová, Iva (advisor)
The aim of this bachelor thesis was study on gene expression in bacterium Cupriavidus necator H16 that is known as a model bacterium for the metabolism of polyhydroxyalkanoates. In the first part of this thesis, the optimalization of RT-qPCR method was performed. The optimized method was implemented on the study on gene expression. Furthermore, there were tested several commercial isolation kits for the genomic DNA isolation, the RNA isolation and the reverse transcription of the RNA and synthesis of the complementary DNA. These kits were compared in order to choose the one that would have provided the most relevant results and also the kit handling would have been simple and safe. There were different results accomplished for all kits. This means the kit used for the isolation had unneglectable impact on the quality of the isolated nucleic acid and therefore also on the success of the whole measurement. Isolated genomic DNA was used for optimalization and calibration. Isolated RNA and complementary DNA were used in the second part of the thesis. In this part, the studied bacterium was cultivated under various conditions and carbon sources. Subsequently, the optimized RT-qPCR method was performed and used for study on gene expression of chosen genes involved in the biosynthesis of polyhydroxyalkanoates. There were more significant differences in gene expression observed for fructose as a carbon source, compared to -butyrolacton as a carbon source. The greatest increase of the gene expression for fructose as a carbon source was measured for gene encoding 4-hydroxyphenylacetate-3-hydroxylase. There were more considerable differences in gene expression observed for -butyrolacton as a carbon source only for gene encoding 4-hydroxybutyrate dehydrogenase. Therefore, the choice of the carbon source impacts fundamentally the gene expression.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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Utilization of spent wood chips for biotechnological production of PHA
Ladický, Peter ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the suitability of wood shavings and sawdust as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most appropriate approach of hydrolysis of wood shaving and sawdust and the effect of polyphenol and furfural concentration on ability to accumulate PHA was studied. Burkholderia sacchari had greater ability to accumulate PHA compared to Burkholderia cepacia. PHB values 87–89 % were achieved when Bulkholderia sacchari was cultivated on medium that contained detoxified hydrolysate of sawdust. Sawdust is therefore a promising substrate for microbial production of PHA in terms of reducing production costs and high content of PHB in biomass.
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