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Modification of porosity of bacterial cellulose in situ
Ondruchová, Barbora ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the production and modification of porosity of bacterial cellulose in situ using the bacterium Komagataeibacter xylinus. The theoretical part of the work was focused on the review of various methods of culturing Komagataeibacter xylinus and the production of porous samples of bacterial cellulose. The sizes of pores in bacterial cellulose depend mainly on the applied cultivation method. Bacterial cellulose produced statically or dynamically contains pores with the dimensions of approximately 0.02 µm to 10 µm. The difference in porosity in bacterial cellulose prepared by static and dynamic cultivation was confirmed experimentally. The production yields of bacterial cellulose were compared and discussed. Next, the porosity of the bacterial cellulose was modified in situ by the addition of wax particles. Scanning electron microscopy confirmed, that the accumulation of wax particles in the production medium could significantly support the porosity of bacterial cellulose and, at the same time, increase its production.
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Biotechnological production of PHB using non-traditional carbon sources
Fialová, Tereza ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
The bachelor thesis was focused on studying the possibilities of using waste rosehip byproducts obtained after the production of liqueurs as secondary carbon sources for the production of poly (3-hydroxybutyrate) (PHB). Cupriavidus necator was used for PHB biosynthesis. All cultures were run as submerged batch cultures in mineral media containing microelements in Erlenmeyer flasks for 72 hours. Rosehip oil obtained by hexane extraction from rosehip seeds and an enzymatic hydrolysate prepared from rosehip waste pulp were used as secondary carbon sources. After 72 hours of fermentation, yields showed that rosehip oil is a comparable carbon source to fructose for culturing of C. necator, in contrast to the enzymatic hydrolysate. Fermentation using 20 g / l rosehip oil produced 10.82 g / l biomass and 6.20 g / l PHB. This is about 21.6% more biomass and 13.1% more PHB than was received by fermentation using 20 g / l fructose. Fermentation of C. necator using an enzymatic hydrolysate with a reducing sugar concentration of 20 g / l gave only 2.92 g / l biomass and 0.12 g / l PHB. Even the combination of enzymatic hydrolysate with fructose did not prove satisfying yields for the production of PHB. Next, in liqueur, rosehip oil and enzymatic hydrolysate was also determined the content of total reducing sugars, phenolics, and total antioxidant activity. Moreover, the fatty acid profile and the content of dyes was determined in rosehip oil. The results showed that all selected rosehip products showed a significant range of total polyphenols (2.04 - 12.4 g GAE /l sample) and antioxidant activity (1.3 - 2.0 mmol TE /l sample).
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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.
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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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Aflatoxins in food and their influence on DNA and cell lines
Šislerová, Lucie ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
Aflatoxins present a great danger due to their high toxicity and carcinogenicity, which is not easily avoided in everyday life. Intoxication with aflatoxins causes a wide range of diseases ranging from mild diseases to organs necrosis or death. Aflatoxins mostly affect the liver, where it degrades and the formation of subsequent metabolites, which are the most toxic to the body. For this reason, their precise determination and understanding of the principle of their effect is very important. In this work, methods for monitoring and closer determination of aflatoxin effects on human cells were calibrated. The methods that were used are: MTT viability assays, fluorescence microscopy and flow cytometry. Next, the amount of aflatoxins present in different foods with different storage conditions was measured. For this analysis were used ELISA assays RIDASCREEN Aflatoxin Total and RIDA Aflatoxin column. Calibrated methods were compared with the methods already used to determine the effect of aflatoxins and the results of the ELISA tests were compared with the limits of aflatoxin levels permitted by the Czech legislation. None of the controlled foods contained above-the-limit concentration of aflatoxins, which in the Czech Republic is set at 4-10 µg/l (varies for different types of food). Foods that were poorly stored but not visibly affected by fungi showed the highest levels of aflatoxins. The LD50 value for aflatoxin B1 was determined to 12,25 µM. The type of cell death caused by aflatoxins was determined by flow cytometry and these data were further confirmed by fluorescence microscopy images.
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Preparation of DNA with local DNA structures
Lofítková, Ellen ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
In this thesis I focused on quadruplexes and cruciforms formed by DNA. Plasmids pBluescript and others derived from it by inserting oligonucleotid sequences were studied. Sequences forming quadruplexes and cruciforms were found by in silico analysis by QGRS Mapper and Palindrome analyser. Plasmids were transformed into E. coli, isolated and then cleaved with enzymes S1 nuclease and restriction endonuclease ScaI. Cleaving with S1 nuclease predicated presence of local structure. Combined S1/ScaI cleavage did not bring satisfying results due to lost of DNA during purification.
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Influence of oxidative pressure on bacterial cells
Dugová, Hana ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the impact of oxidative pressure on the bacterial strain Cupriavidus necator in its two forms: Cupriavidus necator H16 producing PHB granules and Cupriavidus necator PHB-4 as a mutant that is not capable of producing granules. The thesis compares different influences causing oxidative stress that is demonstrated by the occurrence of ROS in the cell. Furthermore, the cells were analysed with a flow cytometer and a fluorescence microscope. During the analysis it was necessary to use different types of fluorescence probes. The oxidative stress was created by means of hydrogen peroxide at various concentrations. Further tests focused on Fenton’s reaction including ammonium iron sulfate and cobalt chloride hexahydrate. Propidium iodide as a fluorescence probe was used to determine the viability of the bacterial cells, and BODIPY was tested as a lipophilic dye. Finally, the ROS in the cell was detected by H2DCFDA and CM–H2DCFDA, fluorescence probes that had to be optimised for the respective technique. The result of this bachelor thesis is the confirmation of the hypothesis that PHA granules production increase the resistance of Cupriavidus necator against oxidative stress.
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Biotechnological production of selected biopolymers employing Haloferax mediterranei
Strečanská, Paulína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is to study of production of selected biopolymers, polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by Haloferax mediterranei. The first part contains theoretical background including properties of biopolymers, advantages and purposes of commercial production, characteristics and positive aspects of cultivation of extreme halophile archaea Haloferax mediterranei. The experimental part is focused on study of effects of concentration of waste substrates, such as proteolytic whey hydrolysate and feather hydrolysate on PHA and EPS production. The effect of medium volume and its correlation with acceess of oxygen on biopolymer production were studied as well. PHA, in particul copolymer P(3HB-co/3HV) was analysed by gas chromatography with FID detection. The content of 3HV in PHA to 15,09 % was achieved using medium which contains 25 % proteolytic whey hydrolysate without glucose supplementation. Medium with 10 % of feather hydrolysate and 10 % of feather hydrolysate with 30 g/l glucose proved to be suitable in volume 50 ml in 250 ml Erlenmeyer flask. Haloferax mediterranei was able to produce biomass and accumulate PHA using protein substrate without need of saccharide supplementation.
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Determination of biological activity of adiponectin, a recombinant protein using cell culture
Pernicová, Iva ; Rittich, Bohuslav (referee) ; Ševčík, Mojmír (advisor)
This thesis deals with the determination of biological activity of adiponectin, a recombinant protein using cell culture. First it was important to acquire the working skills for the cell culture of cell line 3T3-L1. An optimal concentration of inactivated fetal bovine serum in cell culture media was determined. A stimulation of the cell proliferation by HB-EGF, PDGF-BB and bFGF growth factors was observed at various concentration levels. Afterwards the biological activity of adiponectin was determined as an inhibition of growth stimulation with 5 ng/ml PDGF-BB. This biological activity assay for adiponectin was also conducted with lyophilized adiponectin and a growth factor bFGF (0.1 ng/ml). The lyophilization did not affect the biological activity of adiponectin.
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The study of enzyme activities in compost
Pernicová, Iva ; doc.Mgr.Pavlína Pelcová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
This thesis deals with the determination of enzyme activities in compost for consideration their changes during the composting. The changes in the activity of particular enzymes serve as the indicators of the activity of microorganisms, which are found in the compost. In the practical part of the work the changes in activity of proteases, cellulases, lipases, dehydrogenases and ureases within 21 days of composting under laboratory conditions were determined. The change in pH was observed as well. The changes in the pH-values and all enzyme activities except ureases present in the first week the same trend associated with the use of available substrates. The changes in the activities of the key enzymes in compost under laboratory conditions were compared with the changes in activities in compost under real conditions, which is the indicator of the different composting process under the given circumstances.
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