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Influence of plasma activated water on fungal diseases
Moskvina, Anastasia ; Krčma, František (referee) ; Kozáková, Zdenka (advisor)
This bachelor thesis focuses on influence of plasma treated water on fungal diseases. The theoretical part is dedicated to plasma-liquid interactions, properties and application of plasma activated water and its influence on different microorganisms. Plasma activated water contains reactive oxygen species which cause inactivation of living cells, making it a potential sterilizer. The experimental part of this work compares the effectiveness of three plasma treated water preparation techniques. Dielectric barrier discharge system was used to activate water above its surface. For the under the surface activation, a two-electrode system of both alternating and direct current was used. Plasma treated water was then used to prepare a suspended mixture with Aspergillus niger spores. The latter was then cultivated on agar plates for 72 hours. The overall effect was evaluated in amounts of colony forming units. The experimentally obtained data was processed and discussed in the results and discussion section of the thesis. It was found that the underwater surface activation had more significant effect on mold deactivation, which corresponds with the information contained in the theoretical part. Although the effect of dielectric barrier discharge was not as promising, all three methods used led to a decrease of colony forming units in comparison to the non-treated control sample.
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Influence of plasma and plasma activated water on fungi Aspergillus niger
Žitný, Michal ; Čechová, Ludmila (referee) ; Kozáková, Zdenka (advisor)
The thesis deals with the issue of decontamination of Aspergillus niger mold using plasma, plasma-activated water and their combination. The theoretical part concerns the effects on the general properties of plasma, its generation, effects on microorganisms known so far and its use in industry with a special focus on medicine. It also focuses on the generation and characterization of plasma-activated water, its effects on bacteria, yeasts and fungi and its use. It also deals with the description of fungi, their occurrence and their sterilization. Part of the theory was aimed directly at the fungus Aspergillus niger. Its cultivation and its industrial uses, such as its use for the cultivation of citric acid, were described here. The experimental part deals with the characterization of plasma-activated water generated using a torch jet. The influence of the supplied power on the formation of particles in plasma-activated water was monitored. The decontamination effect of the plasma, generated by the torch jet, alone and in combination with plasma-activated water generated by alternating and direct current is monitored. The characterization of plasma activated water was performed with 20 ml of distilled water at a carrier gas rate of 2 l/min and a power of a microwave source of 9 and 12 W. The decontamination effect of plasma and its combination with plasma-activated water was observed by inoculating 100 l of a ten-fold and a hundred-fold diluted treated mold culture on Petri dishes with malt agar. The evaluation of the effect was carried out by counting the grown fungal colonies and subsequent comparison with control samples. All measured and obtained data were processed in the results. Plasma activated water generated by the torch jet has been found to contain higher concentrations of nitrogenous substances and lower concentrations of hydrogen peroxide, with the generated nitrate concentrations being higher at lower output than at higher output. DC-generated plasma activated water containing low concentrations of nitrogenous species and a very high concentration of hydrogen peroxide compared to PAW generated by a torch jet. Furthermore, plasma-activated water generated using a high frequency source contains minimal concentrations of nitrogenous substances and slightly elevated concentration of hydrogen peroxide in regard to torch jet PAW. The highest decontamination effect of all the methods used was the combination of plasma and plasma activated water, prepared by a plasma nozzle using direct voltage, when the plasma was applied first. On the contrary, the least effective method was the application of plasma and plasma activated water, generated by high frequency voltage. Limiting oxygen access had almost no effect on direct plasma treatment, but other methods were significantly affected by it. The greatest difference in decontamination effect was observed for the combination of plasma and plasma activated water, generated by high frequency current, where the difference was up to 30 %, but all methods had a significant decontamination effect compared to the control.
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Preparation and characterization of cyanide hydratase from Aspergillus niger and nitrilase from Arthroderma benhamiae
Hradilová, Iveta ; Vaněk, Ondřej (advisor) ; Martínek, Václav (referee)
Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)
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Preparation and characterization of cyanide hydratase from Aspergillus niger and nitrilase from Arthroderma benhamiae
Hradilová, Iveta ; Vaněk, Ondřej (advisor) ; Martínek, Václav (referee)
Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)
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Waste materials utilization for preparing hydrolysates for the fermentation phase.
Vadovičová, Natália ; Hrstka, Miroslav (referee) ; Babák, Libor (advisor)
Bachelor thesis focuses on the study and comparison of different types of hydrolysis, their optimization and maximization of yields for the upcoming fermentation. Orange peel was chosen as a substrate to conduct the experiments. First, the substrate was mechanically grinded to form a suspension. Each suspension then underwent one out of the examined methods of hydrolysis. Chosen methods were physical, such as microwaves, increased temperature or ultrasound, and chemical acidic and alkaline. Combinations of both types were also examined. The last optimized method was enzymatic hydrolysis. First set of experiments was conducted using enzymes Novozymes® NS50013 and NS50010. Production of cellulase and pectinase enzymes by A. niger during solid-state fermentation that lasted 10 days was also studied. The yields of reducing sugars of all the experiments were calculated using the Somogyi-Nelson method. Enzymatic hydrolysis was proven to be the most effective using the combination of both of the enzymes for a period of 96 hours at pH = 4.5 and temperature 45 °C. Yield of the reducing sugars under these conditions reached 27,4241 ± 0,0007 gl-1.
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Study of Sterilization Effects Initiated by Dielectric Barriere Discharge
Slámová, Jitka ; Pekárek, Stanislav (referee) ; doc. RNDr. Karol Hensel, Ph.D., oponent (referee) ; Krčma, František (advisor)
The overall goal of the presented dissertation thesis was to study the sterilization efficiency of dielectric barrier discharge operated at atmospheric pressure. The fungi Aspergillus niger, gram-positive bacteria Bacillus subtilis and in some experiments also gram-negative bacteria Escherichia coli were used as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. The samples of microorganism were placed on paper Whatman 1 or PET foil and exposed to plasma. The plasma was generated in argon, nitrogen, synthetic dry/humid air with frequency up to 10 kHz and plasma power density in the range of 1,2-2,9 W/cm3 (according to the process gas). The influence of process gas, plasma power density, plasma exposition time, type of microorganism and material of the substrate on the sterilization effect of dielectric barrier discharge was evaluated. Furthermore the contribution of each single mechanism (UV radiation, temperature and reactive species) to the sterilization effect of plasma and influence of gas humidity was evaluated. The DBD was analysed by means of optical emission spectroscopy, thermocouple was used to measure temperature during a sterilization process. In order to verify the mechanical damage of the microbial cell or the substrates during the plasma process the samples were studied by scanning electron microscopy. Generally, on the basis of experimental results, at increasing treatment times, the remaining number of spores (CFU) decreased. Similarly at increasing the plasma power input, the sterilization rate increased. When sterilising the spores of A. niger in plasma using different process gasses, the efficiency of plasma sterilization decreased as follows: argon, humid synthetic air, nitrogen and dry synthetic air. The results observed in argon plasma using different microorganism demonstrated that the sensitivity of vegetative cells resp. spores to DBD decreased as follows: A. niger spores, B. subtilis vegetative cells, E. coli vegetative cells and B. subtilis spores. Simultaneously results observed for sterilization of spores and vegetative cells of B. subtilis and A. niger demonstrated that the spores are generally more resistant to plasma than are the corresponding vegetative cells. Combining the results of contribution of each single mechanism, optical emission spectroscopy and inactivation characteristic it was found out that the reactive species significantly contribute to the plasma sterilization in all process gasses. Furthermore the inactivation process can be partly assisted by UV radiation and also the temperature can contribute in limited extent to inactivation process in some gasses. The contribution of UV radiation to the plasma sterilization decreased as follows: nitrogen, argon, dry syntetic air and humid syntetic air. Moreover it was found out that the contribution of each single mechanism can be species dependent, this is due to the different response of microorganism to the unfavorable external conditions. SEM analysis of the substrates prooved the etching actions of the plasma generated in all process gasses on the surface of the PET foil. The several minute plasma exposition of the PET foil resulted in the occurence of the „hole corrosion“ on the PET surface. Contrary to these there were no visible changes observed in the paper structure.
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Influence of humidity on total sterilisation effect of dielectric barrier discharge
Kramárová, Petra ; Bartlová, Milada (referee) ; Kozáková, Zdenka (advisor)
The main subject of this diploma thesis is the study of the effect of humidity on the total sterilization effect of the dielectric barrier discharge. Sterilization is a process which can eliminate all forms of life. The plasma sterilization is one of the methods that are suitable for sterilization of temperature and chemical sensitive materials. This sterilization method was proved to be effective on the wide spectrum of procaryotic and eucaryotic microorganisms. Basically, the main inactivation factors for cells exposed to plasma are heat, UV radiation and various reactive species. Dielectric barrier discharge (DBD) operating at atmospheric pressure was used for the sterilization of the samples. The discharge was generated in dry air and in humid air. The plasma power densities were 2 160 mW.cm-3, 2 279 mW.cm-3 and 2 760 mW.cm-3 (dry air) or 2 326 mW.cm-3 and 2 850 mW.cm-3 (humid air). Humidity of air was achieved using a wash bottle filled with water through which air flowed into the DBD reactor. Fungi spores of Aspergillus niger were used as model microorganisms. Whatman paper No. 1 was used as the carrying medium. When comparing sterilization efficiency of humid and dry air operating at the same conditions, the higher sterilization effect was observed in humid air. The sterilization effect of the DBD generated in air was compared with results obtained during plasma generation in argon and nitrogen. At the same conditions, the highest sterilization effect was observed in argon, followed by humid air, nitrogen and dry air. It was found out that in our experimental setup the active species are probably the main inactivation mechanism. The influence of temperature on the inactivation of microorganisms was completely negligible. The discharge parameters were studied by means of the optical emission spectroscopy (OES). Plasma treated samples were analyzed employing scanning electron microscopy (SEM). Damage of the microorganisms due to the effect of plasma as well as plasma effect on the structure of the carrying medium was evaluated.
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The employment of waste paper to microbial production of cellulases
Peterek, Miroslav ; Flodrová, Dana (referee) ; Omelková, Jiřina (advisor)
Study of the employment of waste paper to microbial production of cellulase was carried out using Aspergillus niger cultivation on carbon sources that have been waste office paper and cardboard, humidified by no – carbon medium or distiled water. Cultivation took place in the SSF way in Erlenmeyer flasks and columns. Concentration of extracellular proteins, cellulase and protease activity for selected samples were monitored. It was found that the most advantageous method of cultivation in terms of cellulase activity production is the cultivation in the column washing by no – carbon medium in three day intervals.
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The study of production of hydrolytic enzymes for cellulose wastes treatment
Řezáčová, Barbora ; Flodrová, Dana (referee) ; Omelková, Jiřina (advisor)
The study of production of hydrolytic enzymes dealt with the production of cellulase and polygalacturonase by two microbial strains - Aspergillus niger and Aureobasidium pullulans. The enzymes were produced in solid-state fermentation system. The wheat straw and sugar beet pulp were used as a substrate. The substrates were moistened by water, mineral solution or by medium with glucose. The effect of mineral solution and glucose on production of these enzymes were monitored during cultivation. The highest production of polygalacturonase was achieved by Aspergillus niger during cultivation on sugar beet pulp moistened by mineral solution. The highest production of cellulase was achieved by Aspergillus niger during cultivation on wheat straw moistened by medium with glucose.
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Study of elementary inactivation processes acting during sterilization of eucaryotic systems in dielectric barrier discharge
Vojkovská, Hana ; Machala, Zdenko (referee) ; Kozáková, Zdenka (advisor)
This diploma thesis is focused on studying of the effect of the dielectric barrier discharge (DBD) on eucaryotic microorganisms. Plasma sterilization is considered to be an alternative method to conventional sterilization processes. Contrary to standard decontamination methods it doesn´t stress exposed material by heat, pressure and chemicals. Plasma acts on eucaryotic and procaryotic systems by means of synergy of three inactivation mechanisms. They are various reactive species, UV radiation and heat. The Aspergillus niger has been chosen as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. Plasma was generated in dielectric barrier discharge (DBD) at atmospheric pressure. Nitrogen and argon were used as working gases, paper and PET foil were used as carrying media. The influence of various working conditions on the sterilization effect was studied. Namely it was the influence of plasma exposition time, plasma power density, the type of operating gas and type of supporting medium. The effect of UV radiation in combination with temperature, temperature and direct plasma were studied separately. According to our results the efficiency of DBD increases with plasma power density, resp. plasma exposition time. When comparing sterilization efficiency of nitrogen and argon operating at the same conditions, the higher sterilization effect was observed in argon. The influence of the carrying medium on sterilization effectiveness was proved. It was caused by the different structure of surface. It was found out, that in our experimental setup the active species are probably the main inactivation mechanism. The influence of temperature on inactivation of microorganisms was negligible. The combination of UV radiation and temperature reached the decontamination level about 2 orders. The discharge parameters were studied by means of optical emmision spectroscopy. Scanning electron microscopy enabled to evaluate possible damage of exposed materials through DBD.
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