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Study of the production of polyhydroxyalkanoates using selected thermophilic representatives of the genus Aneurinibacillus
Řeháková, Veronika ; Kovalčík, Adriána (referee) ; Pernicová, Iva (advisor)
The subject of the diploma thesis is the production of PHA copolymers by thermophilic Gram-positive bacteria of the genus Aneurinibacillus. The theoretical part is devoted to the general characterization of PHA materials, their properties, use, and also to the production of PHA polymers by these bacteria. The experimental part deals with the production of PHA copolymers using selected thermophilic members of the genus Aneurinibacillus. Firstly, the specific enzyme activity of PHA synthases was determined, and then the production of PHA copolymers was screened using selected lactones (-valerolactone, -hexalactone, -valerolactone) and diols (1,6-hexanediol, 2,3- butanediol and 1,4-butanediol). These experiments were performed with six thermophilic producers by the members of the genus Aneurinibacillus, which were isolated from compost and activated sludge. Tested microbial strains have demonstrated the ability to integrate interesting monomers into the PHA structure, including 4-hydroxyvalerate (represented up to 69.3% of the total PHA), 5-hydroxyvalerate (up to 47.1%), or 4-hydroxyhexanoate (up to 31.9%). Subsequently, a closer characterization of the obtained PHA (which were gained by the production of the best producers) using advanced methods (DSC, SEC-MALS, FT-IR) was performed. Finally, the screening of the PHA copolymers production was performed in laboratory bioreactors.
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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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Thermophilic microorganisms application to biodegradation
Varmužová, Tamara ; Márová, Ivana (referee) ; Babák, Libor (advisor)
This thesis is focused on study of biodegradability of modified polyurethane elastomeric films in synthetic medium with minerals and vitamins on tempered shaker by mixed thermophilic aerobic bacterial culture Bacillus and Thermus genera. In most cases addition of all used fillers (carboxymethyl cellulose, hydroxyethyl cellulose, acetylated cellulose, acetylated starch and glutein) led to increased biodegradability of elastomeric films with modifying agent in comparison with elastomeric films without modifying agent (referential). The growth of cultures was strongly increased in presence of elastomeric films modified by 10 % acetylated cellulose and 10 % carboxymethyl cellulose. Elastomeric film biodegradation mechanism included probably two stages: abiotic destruction of elastomeric films and consequent utilization of degradation products by bacterial culture.
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Raman spectroscopy of prokaryotic cells
Večeríková, Paula ; Bernatová,, Silvie (referee) ; Samek, Ota (advisor)
The presented bachelor‘s thesis deals with the Raman spectroscopy of bacterial cells. This method proves to be very suitable for monitoring the culture conditions of these bacterial strains because it is essentially non-destructive and fast compared to gas chromatography. Due to it‘s non-destructive nature, it is possible to further cultivate the measured cells. This analytical method can also be used to sort and select individual cells with increased ability to produce PHB. This would enable the possibility to select the generation of so-called PHB superproducers. Polyhydroxybutyrate (PHB) belongs to the group of polyhydroxyalkanoates (PHA), which serve as a substitute for plastics made in the petrochemical industry, whose consumption is increasing, especially during a pandemic. Current biodegradable substitutes are based on starch, which, in turn, depletes nutritional material for humans and animals. This bachelor thesis can serve as a basis outline for reducing the cost of PHB production, because Raman spectroscopy can be used in cell culture as a sensor of PHB content in response to a bacterial strain. In the experimental part of the work, the Raman spectra of selected bacteria were measured, where the result showed the greatest response to PHB in Chelatococcus shambunathi and the lowest in the thermophilic isolate BZ (Paenibacillus sp.). Quantitative information on the content of PHA in bacteria was obtained by gas chromatography, where the highest content was the bacteria- Chelatococcus shambunathi and the lowest Termobacillus composti. The subject of the second part was the separation of a mixture of PHA-producing and non-PHA-producing bacterial cultures. The result clearly proves that the mixture is separable based on the intensity of the selected sorting parameter.
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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.
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Complementary analysis of procaryotic cells by electron microscopy and Raman spectroscopy
Ikrényiová, Terézia ; Sedláček, Petr (referee) ; Hrubanová, Kamila (advisor)
This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.
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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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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Biotransformation of ferulic acid to sensory active compounds
Opial, Tomáš ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotransformation of ferulic acid to sensory active substances. The experiment was based on monitoring the biotransformation potential of selected microorganisms by high performance liquid chromatography (HPLC) method while the cultivation was performed in two parallel runs and samples with added ferulic acid were divided into 24-hour effect and 48-hour effect of ferulic acid on bacterial cultures. Thermophilic bacteria and halophilic bacterium Halomonas neptunia have been found to be the most suitable candidates for the biotransformation of ferulic acid to sensory active substances. In both samples of Schlegelella thermodepolymerans (DSM 15344 and DSM 15264) was formed 33 mg/l and 76 mg/l of vanillic acid and 81 mg/l and 71 mg/l of 4-vinylguaiacol after 24 hours of ferulic acid effect. In a sample with T. taiwanensis was formed 61 mg/l of vanillic acid and 32 mg/l of 4-vinylguaiacol after 48 hours of ferulic acid effect, and in a sample of R. xylanophilus was formed 56 mg/l of 4-vinylguaiacol. In the sample with H. neptunia after 24 hours of ferulic acid effect was formed 296 mg/l of vanillic acid, which was up to 59% of the conversion of the added ferulic acid and in a sample with H. organivorans after 24 hours of ferulic acid effect was formed 71 mg/l of vanillic acid. However, after 48 hours of ferulic acid effect, vanillic acid degraded. In addition to screening of biotransformation potential, the sequence of bacterial enzymes, involved in biotransformation, was also compared with protein sequences in the database using the BLAST search tool. The presence of genes encoding enzymes involved in biotransformation has been demonstrated for almost all used microorganisms except H. neptunia, for which no gene has been identified. The highest number of genes present in bacteria was with the enzymes feruloyl-CoA synthetase, enoyl-CoA hydratase/isomerase, acetoacetyl-CoA thiolase and vanillin dehydrogenase.
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Screening of biosurfactant production in selected thermophilic bacteria
Řeháková, Veronika ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
The subject of the bachelor thesis is the production of biosurfactants by selected thermophilic bacteria. The theoretical part contains a general characterization of thermophilic bacteria, describes the structures and classification of biosurfactants, characterizes their properties and uses. The final part of the theoretical part deals with the production of biosurfactants by thermophilic bacteria. The experimental part deals with the screening of biosurfactant production by selected thermophilic bacteria. Thermophilic bacteria were obtained from composts and activated sludges. Isolation is not a subject of the thesis. In total 39 bacterial isolates were cultivated and their supernatants were tested by screening methods – emulsification capacity assay and solubilization of crystalline anthracene. Based on the results of these methods, 14 most promising producers were selected. These bacterial strains were re-cultured to obtain a new set of supernatants. For more detailed screening of production by selected thermophilic bacteria, in addition to determining the activity of the emulsion and solubilization of crystalline anthracene, other methods were added: direct surface tension measurement – Du-Noüy-Ring, drop pouring method and oil spreading method. The results were evaluated and the measurements were repeated for a new set of supernatants to verify the reproducibility of the production. The results of all the tests were compared and then three most reliable thermophilic bacterial producers were determined.
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