|
|
Controlled Production and Degradation of Selected Biomaterials
Obruča, Stanislav ; Demnerová, Kateřina (referee) ; Vávrová, Milada (referee) ; Němec, Miroslav (referee) ; Márová, Ivana (advisor)
Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.
|
|
|
Plasticizing of polyhydroxyalkanoates in microbial cells - inspiration for development of artificial controlled release systems
Liczka, Jan ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
The diploma thesis is focused on the study of the mechanism of plasticization of polyhydroxyalkanoates (PHA) in vivo, focusing mainly on methods of isolation native PHA granules and physical and chemical methods of initiation their phase transitions. The literature search prepared in this work deals with this microbial polyester and further focuses on individual methods of isolating PHA from bacterial cells. The main task of the experimental work was to optimize the isolation process of native PHA granules, as well as to design and test processes that initiate the PHA crystallization in isolated granules, as well as analytical procedures to correctly detect this phase transition. Isolation of PHA granules from Cupriavidus necator was performed by enzymes, centrifugation several times and sonication of the cells. Fourier transform infrared spectroscopy (FTIR) and an enzymatic method in which amorphous PHA is selectively degraded by PHA-depolymerase with turbidimetric detection of its loss in dispersion was used to detect PHA crystallization. Techniques based on fluorescence staining of granules (fluorescence spectroscopy and flow cytometry) were used in a more detailed study of the mechanism of PHA plasticization in granules. The results of the analyzes confirmed, among other things, that the optimized procedure makes it possible to isolate granules in the amorphous state, which remain amorphous even after drying and are comparable to native granules in their basic physicochemical properties.
|
|
|
Use of mealworm (Tenebrio molitor) for isolation of polyhydroxyalkanoates from bacterial biomass and simultaneous production of food
Najbrtová, Johana ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the use of mealworms (Tenebrio molitor) to isolate polyhydroxyalkanoates from bacterial biomass and current food production. Cupravidus necator H16 and Schlegelella thermodepolymerans were used to prepare bacterial biomass containing polyhydroxyalkanoates (PHA). The ability of mealworms to isolate PHA from bacterial biomass of various types was determined. First, the content of PHA and crude proteins in individual types of biomasses was determined. Subsequently, two biological isolation experiments were performed, in both cases the results were compared with a control sample that consumed bran. The conversion of feed into the form of fecal pellets was monitored, where the best results were obtained from a sample of mealworms fed with dried biomass C. necator H16. Furthermore, the weight loss during the experiment, the percentage of survivors, the amount of feed not consumed, and the number of pupae were evaluated. The fecal pellets obtained by biological isolation had a low PHA content and were therefore subjected to purification. The reagent with the best results was 0,1 M NaOH. Another part of this work was to determine the crude protein content of mealworm flour made from mealworms from experiment two. The sample fed with biomass produced by Schlegelella thermodepolymerans had the highest protein content.
|
|
|
Polyhydroxyalkanoates as metabolites of prokaryotic microorganisms adapted to extreme conditions
Vodička, Juraj ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The objective of this work is a study of polyhydroxyalkanoates as bacterial carbon and energy storage forms, which are produced intracellularly. The theoretical part of the study is focused on physical properties of PHA, their biotechnological production and degradation. The experimental part deals with production of polyhydroxyalkanoates by halophile bacterium Halomonas organivorans using several different carbon substrates. Firstly, the gene encoding for PHA-synthase, phaC, was successfully amplified and detected. Extracellular lipolytical activity was also proved. Secondly, the most appropriate substrate for bacterial growth and PHA production were found to be hydrocarbons, especially galactose and mannose. Due to these results, it is highly desirable to use coffee grounds for the biotechnological production of PHB, as galactomannan-rich waste. On the other hand, lactose and xylose have shown to be unsuitable carbon sources. For that reason, waste whey and lignocellulosic materials rich for pentoses are inconvenient substrates for the intended purpose. There is a potential use of waste molasses because of good results of sucrose at contrast. By the cultivation on kitchen waste and waste glycerol were the growth slow and the production of PHA reduced significantly. The optimal salt concentration for bacterial growth and PHA production were discovered as 60 grams per litre. Addition of several precursors of 3-hydroxyvalerate after 24 hours of cultivation have not induced the copolymer P(3HB-co-3HV) formation. Even though these precursors have inhibited the bacterial growth, the significant increase of the PHB percent contend was observed. In general, the yield of the production has been considerable, it should be noted than almost in each successful cultivation the PHA content in bacterial cells exceeded 50 weight %. With a respect to results, more experiments for full evaluation of H. organivorans as a potential biotechnological PHA producing bacteria are regarded.
|
|
|
Development of microbioreactor for microbial biotechnology
Jakešová, Michaela ; Szotkowski, Martin (referee) ; Krejčí,, Jan (advisor)
This master thesis deals with the development of a minibioreactor for microbial biotechnologies. The AlgaTox system – an analytical photobioreactor from BVT Technologies – was the default unit of the new device. The working volume of the new minibioreactor is in the range of 4 to 8 ml. The minibioreactor was composed of a minithermostat, a reaction vessel, an oxygen electrode, a temperature and pH probe, accessories for the supply of air to the liquid and an insertion for the transport of liquid from / to the reactor. The functionality of the assembled device and its characteristics were measured. Furthermore, an operating procedure for the decontamination process using a hydrogen peroxide mist was developed for the new equipment. An operating procedure for culturing microorganisms in a minibioreactor was also set up. Pilot cultivations of Halomonas halophila were demonstrated in the prepared equipment. In these cultivations, three assemblies for air supply to the liquid were tested. However, none of the assemblies was able to provide a sufficient supply of oxygen to cell culture – the dissolved oxygen value always dropped to 0%. For the further development, a new assembly was designed for the supply of air to the liquid - an aeration ring from a membrane tube.
|
|
|
Photoprotective properties of PHB granules
Hesko, Ondrej ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The objective of this work is study and design of physical-chemical methods for verification of photoprotective properties PHB inclusions using bacteria Cupriavidus necator H16 and its mutated bacteria strain Cupriavidus necator PHB-4 which differs in production capability of polyhydroxyalkanoates. Basic information about of polymer inslusions and methods used for proving photoprotectivity are discussed in theoretical part. Methods such as nephelometry and continous fluorescence microphotolysis (CMF), which is used to characterize the mechanism of interaction of light with inclusions, were optimized. In experimental part properties of light scattering in bacteria were determined and characteristic curves were obtained. Further the photostability of fluorescence probe specific for nucleic acids were compared in bacteria strains. This method should prove bigger tolerance of bacteria H16 for UV light. It should also define possible interaction of nucleic acids with PHB inclusions.
|
|
|
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.
|
|
|
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.
|
|
|
The effect of magnetic field and other selected stressors on physiology of bacterial cells
Mrázová, Kateřina ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the effect of magnetic field and organic substances, namely benzene and p-nitrophenol, on cell of PHA producing bacteria Cupriavidus necator H16 and mutant strain Cupriavidus necator PHB4, which does not produce polyhydroxyalkanoates. Static magnetic field was generated by both permanent magnet and electromagnet. The effect of magnetic field on the growth of bacterial cells was studied using growth curves. It was found that cultivation in magnetic field and mineral medium mostly inhibits bacterial growth. Also the amount of polyhydroxyalkanoates was observed using FT-IR, flow cytometry and microscopy with fluorescent dye. Growth curves and flow cytometry were also used to study the influence of organic substances on bacterial cells. It was found that while benzene does not affect either C. necator H16 or C. necator PHB4, p-nitrophenol acts as the inhibitor of bacterial growth for both cultures. Finally the impact of p-nitrophenol on the accumulation of PHA was studied using gas chromatography.
|
|
|
Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.
|
guest ::
