|
|
Biotechnological production of PHA by selected bacterial isolates
Matějka, Filip ; Šedrlová, Zuzana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the production of polyhydroxyalkanoates (PHA) using thermophilic bacterial isolates with designation 34, 35 and BŽ. Bacterial straines were isolated from activated sludge and compost The theoretical part contains a description of PHA, extremophilic bacteria and detection techniques for the determination of polyhydroxyalkanoates. In the experimental part, the presence of the phaC gene, which is crucial for the ability to produce PHA, was first determined by PCR and bacterial strains were also subjected to DNA sequencing of 16S rRNA gene which enabled preliminary taxonomical classification of the isolates. In the next part of the experimental work, the conditions for biomass growth and PHA production were optimized. Suitable carbon source, the ideal temperature for cultivation and the influence of precursors on the production of copolymers were studied and identified. The composition and proportion of PHA were determined spectrophotometrically and by GC-FID. Finally, visual screening of PHA accumulation inside bacterial cells was performed using fluorescence microscopy.
|
|
|
Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
|
|
|
Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
|
|
|
Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
|
|
|
Analysis of mycotoxins in barley and malt
Čuta, Robert ; Obruča, Stanislav (referee) ; Běláková, Sylvie (advisor)
This bachelor´s thesis deals with the mycotoxines present in cereals, especially in barley and malt. Teoretical part summarizes the knowledge of mycotoxines and their occurrence. The possibilities of mycotoxines determination are held forth in this thesis. In the experimental part an ultrasonic extraction metod of deoxynivalenol was optimized. For the deoxynivalenol determination was used a method of high-performance liquid chromatography with a mass detector (HPLC – MS/MS). This method was used for deoxynivalenol determination in barley and malt produced from this barley. The bachelor´s thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.
|
|
|
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.
|
|
|
Role of extracellular DNA in microbial biofilms
Gromov, Anton ; Chytilová, Aneta (referee) ; Obruča, Stanislav (advisor)
Abstract This bachelor’s thesis deals with defining the role of extracellular DNA in microbial biofilms. The thesis includes a part about the basic characteristics and functions of biofilms, as well as the characteristics and functions of extracellular DNA. The theoretic part additionally deals with the process of formation of biofilms, and the influence of different types of stresses on it. Also, the paper discusses the role of such stresses. The role of biofilm in bacteria and their reproduction methods are also discussed. An interesting section of the thesis outlines the impact of antibiotics on biofilms, or the ways to gain DNA or use DNase. The eDNA functions and their effects on bacterial defence will be discussed thoroughly. In the experimental part, the biofilm content was examined and compared with cells growing in a liquid and solid medium. Measurement of DNA content in media was carried out and the effects of salt and temperature stresses on biofilm were also measured. Stress conditions affected the production of PHB, which is used as a reserve form of energy and carbon. Total sugars were measured using an anthracite reagent to study the effects of stresses on the content of macromolecules. The presence of eDNA in biofilms and planktonic cells was firstly checked using gel electrophoresis and then accurate concentrations in planktonic and biofilm cells were determined using different spectrophotometric methods.
|
|
|
Preparation and application particles from copolymer of P(3HB-co-4HB)
Ostanina, Kseniia ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with preparation of particles from poly(3-hydroxybutyráte-co-4-hydroxybutyráte). Copolymer was dissolved in acetone and then the solution had been dosed into the anti-solvents. Acetone and anti-solvent are perfectly miscible. Theoretical part focuses on the properties of the copolymer, its production and potential use in medical applications. In the experimental part the copolymer was dripped into selected anti-solvents by syringe and pipette above and below the surface of anti-solvent. The following anti-solvent were chosen: water, methanol, dimethylsulfoxid (DMSO), 50 % acetone-water, 75% acetone-water, 50 % methanol-water, 75 % methanol-water mixtures. There were three copolymers used with different compositions of 4HB units (63 %, 87 % a 33 %). Copolymer composition was analyzed by gas chromatography. Copolymers were prepared by bacterial strain Aneurinibacillus sp. H1. Due to the results particles of size approximately 100-500 nm can be prepared by methods described in this work. The produced particles can be successfully used in medical applications.
|
|
|
Controlled prodution of pullulan by yeast-like organism Aureobasidium pulluans
Skoumalová, Petra ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
Bachelor's thesis is focused on study of influence of exogenous stress factors on biomass and pullulan production by microorganism Aureobasidium pullulans. As a part of this work an overview of stress factors, pullulan producers, its structure, function and technological use was introduced. In the experimental part growth characteristics of Aureobasidium pullulans and pullulan production during growth in optimum conditions and under stress were analyzed. The reduced availability of oxygen resulted in a decrease of biomass production accompanied by increased pullulan production. Chemical stress induced by NaCl significantly affected mainly biomass production. The highest production of pullulan was found at 15 g / l of NaCl. Ethanol stress exhibited growth inhibition and at higher concentration also lack of pullulan production. Peroxid stress exhibited no effect on pullulan production. Short-time exposure to low heavy metal concentration (Se(IV), Cr(III)) influenced pullulan production more positively than long-term effect.
|
|
|
Structure and properties of bacterial biofilms
Mrázová, Kateřina ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of basic properties of cells of bacterial biofilm produced by PHA producing bacteria, specifically Burkholderia cepacia and Burkholderia sacchari. Two cultivation approaches were used to compare different types of cells: at first, the cells were cultivated in bioreactor providing both planktonic and biofilm attached cells, secondly, cells grown on Petri dishes were also included in the study. In experimental part the affinity of each type of cells to hydrophobic phase was studied using BATH method. It was found that sessile cells, either biofilm associated cells as well as cells taken from Petri dishes, prefer hydrophobic phases more than planktonic cells. Also size and Zeta potential were measured using Dynamic Light Scattering (DLS). It was found for studied samples that, cells in these samples tend to aggregate and the difference of size between both types of cells occurs only for biofilm of the strain Burkholderia cepacia. Finally the impact of presence various bacterial cells types on surface tension of water as well as amount of PHA cumulated in different types of cells were observed.
|
guest ::
