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Biotechnological production of polyhydroxyalkanoates on wheat bran
Guziurová, Pavlína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The bachelor thesis deals with the biotechnological production of polyhydroxyalkanoates (PHA) from lignocellulosic waste – wheat bran. The aim was to determine whether the hydrolysate from wheat bran subjected to different types of pretreatment could be used as a substrate for the production of PHA using selected bacterial strains. The selected strains were Halomonas organivorans, Halomonas halophila and two strains of Schlegelella thermodepolymerans. Halophilic strains were proved as the best producers and were subsequently used for cultivations. The hydrolysates after neutral pretreatment were utilized by the bacteria most efficiently, due to the lowest content of microbial inhibitors (phenolic substances) where the bacteria produced the most PHA. The highest value of produced PHA was determined on the hydrolysate after neutral pretreatment by using the strain Halomonas organivorans, namely 2,82 g/l. The hydrolysate was also used for the production of lactic acid bacterial strains of Lactobacillus. The highest achieved concentration of produced lactic acid was 16,73 g/l by the Lactobacillus casei strain.
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Study of 3D bacterial cellulose production using banana peels as a carbon source
Netopilík, Tibor ; Pernicová, Iva (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the study of 3D bacterial cellulose production using Komagataeibacter xylinus using banana peels as a carbon source. The theoretical part deals with the comparison of the properties of bacterial cellulose and plant cellulose, different methods of biotechnological production of bacterial cellulose and its use. The aim of the bachelor thesis was to find out whether it is possible to use banana peel as a carbon source for biotechnological production of bacterial cellulose. Banana peels are waste lignocellulosic material produced, for example, in the production of snacks or fruit or dairy beverages in the food industry. HPLC analysis showed that 1 l of hydrolyzate after enzymatic hydrolysis of 100 g of dry banana peels per 1 l of water contained 8.86 g of glucose and 10.46 g of fructose. The hydrolyzate was used as a carbon source for static and dynamic cultivation of Komagataeibacter xylinus. The yields of bacterial cellulose produced by using banana peels or glucose were compared. The use of banana peels hydrolyzate increased the yields of bacterial cellulose about 170 % in the case of static cultivation and about 220 % in the case of dynamic cultivation. Scanning electron micrographs of bacterial cellulose confirmed the morphological differences between bacterial cellulose produced statically and dynamically. Moreover, the morphology of bacterial cellulose was influenced by the kind of used carbon source.
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Employment of bacterium Tepidimonas taiwanensis for production of polyhydroxyalkanoates on waste substrates
Vidláková, Michaela ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on screening PHA production using thermophilic bacterium Tepidimonas taiwanensis and on the study of possible use of grape pomace, molasses and waste paper as a cheap carbon substrate for culturing the characterized bacteria. At first, testing of basic cultivation parameters was performed, including carbon substrate concentration, oxygen availability, ability to utilize nitrogen sources and selected disaccharides. PHA production from waste substrates was tested in three ways. In the first, pre-prepared solids-free hydrolysates from raw materials were used as the carbon source. The second and third procedures were performed by dosing waste materials directly into the mineral media, which differed only in the presence or absence of the enzyme preparation enabling release of fermentable sugars. The most intensive increase in culture and the highest production of PHA was recorded on grape hydrolyzate. The biomass concentration in this sample reached up to 4.8 g/L with a content of 59 % PHA. On the other hand, the addition of grape marc directly to the production medium did not work at all, which was probably due to the presence of a large amount of inhibitory substances from the pomace. The situation was similar with molasses and waste paper, where the bacterium Tepidimonas taiwanensis was able to grow and possibly produce PHA only to a small extent. The work also managed to characterize the effect of temperature and pH on the activity of the enzyme cocktail Viscozyme L and to determine the temperature and pH optimum PHA synthase of the bacterial strain Tepidimonas taiwanensis in the cell lysate.
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Study on biotechnological potential of thermophilic gram-positive bacterium Brevibacillus sp. Bz
Filimonova, Anastasiia ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
Předmětem předložené diplomové práce je studium biotechnologického potenciálu termofilní grampozitivní bakterie Brevibacillus borstelensis BZ. Teoretická část obsahuje obecnou charakterizaci termofilních organismů a jejich termozymů. Popisuje také adaptivní molekulární mechanismy, které zajišťují termostabilitu těchto proteinů. Závěr teoretické části je věnován biodegradaci odpadních substrátů a polymerů na bázi přírodních a fosilních zdrojů. První část experimentální práce se zabývá produkcí hydrolytických enzymů na různých původních zdrojích a odpadních substrátech. Díky intenzivní enzymatické produkci na původních zdrojích je Brevibacillus borstelensis BZ považován za velmi slibného producenta termostabilních enzymů, konkrétně xylanáz a celuláz. Testovaný bakteriální kmen BZ byl schopen produkce termostabilních enzymů i na odpadních substrátech. Na vybraných substrátech kmen BZ přednostně produkoval xylanázy. Díky tomu byla xylanázám věnována zvláštní pozornost, kdy bylo testováno teplotní a pH optimum. Závěrem experimentální práce byla testována schopnost bakterie Brevibacillus borstelensis BZ degradovat vybrané polymery na bázi přírodních a fosilních zdrojů. Kmen BZ poskytl nový pohled na biologický rozklad polyethylentereftalátu (PET), amorfní frakce kyseliny polymléčné (PLA), semikrystalické PLA a polyhydroxyalkanoátů (PHA). Pozorováním povrchu PET fólie skenovacím elektronovým mikroskopem (SEM) se potvrdilo zdrsnění materiálu, přítomnost rýh a naprosté pronikání bakterie skrz fólii. Pokud jde o polymery na bázi přírodních zdrojů, PHA granule byly zcela degradovány. Studiem morfologie povrchu obou zmíněných PLA bylo prokázáno jasné zhoršení jejich struktury přítomností jam a trhlin na povrchu polymerů.
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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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Encapsulation of active substances into nanofibers and possibilities of their application
Procházková, Lucie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
The master thesis was based on the optimization of the production of nanofiber covers and to gaine the product for subsequent functional use. The production of nanofiber covers was made by electrospinning and forcespinning from selected materials. Polyhydroxybutyrate, gelatin, chitosan and alginate were used as starting materials. After successful optimization, these materials were enriched with active ingredients ampicillin and ibuprofen for the functionalized use of covers for more effective wound healing. The theoretical part was focused on the issue of skin, healing processes, types of wounds and nanofibers, the characterization of selected starting materials for the formation of nanofibers was also mentioned. The practical part was based on the lengthy optimization of the preparation of fiber covers and later enriched with active ingredients. Furthermore, combined covers made of different materials with contents of both active ingredients were designed. This was followed by the characterization of all prepared covers from the point of view of stability in the short and long term. The gradual release of active ingredients was determined spectrophotometrically and by hifh performance liquid chromatography. It was also important to determine the antimicrobial activity of selected active substances. At the end of all testing, combined coatings containing both active ingredients were used for safety testing with human keratinocyte cells (HaCaT). Safety testing was based on determining the viability of human cells using the MTT test, to verify the LDH test. A scratch test was also performed, a wound healing test after the application of devised combined covers.
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Preparation and characterization of modern wound covers
Balášová, Patricie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.
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Biotechnological production of PHA copolymers containing 4-hydroxybutyrate
Kovářová, Radka ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
The proposed diploma thesis aims to study the biotechnological production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The subject of the experimental part was first to select a suitable bacterial strain from five selected microorganisms with different carbon precursors applied at various concentrations. The five selected microorganisms used in the experimental part include bacterial strains Cupriavidus malaysiensis DSM 19416, DSM 19379, and DSM 25816. Furthermore, the strain Thermomonas hydrothermalis DSM 14834 and Aneurinibacillus thermoaerophilus H1 CCM 8960. The experiment shows that the most suitable candidate for biotechnological production is the bacterial microorganism Cupriavidus malaysiensis DSM 19379. Finally, the biotechnological production of the copolymer was investigated utilizing a batch cultivation technique in a laboratory bioreactor.
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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 extremozyme production of selected extremophilic PHA producers
Dyagilev, Dmitry ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
This bachelor thesis deals with the screening of the production of extracellular hydrolytic enzymes in thermophilic microorganisms of the genera Aneurinibacillus, Brevibacillus, Chelatococcus, Pseudomonas, Schlegelella, Tepidimonas and Caldimonas. The ability of selected enzymes, namely proteases, lipases, amylases, xylanases, cellulases and pectinases, was tested in the investigated microorganisms. Such testing made it possible to assess in which microorganisms the production of specific enzymes can be observed. Based on the results of the screening, it was found that Schlegelella aquatica LMG 23380, Tepidimonas fonticaldi LMG 26746 and the investigated microorganisms of the genus Chelatococcus did not show the ability to produce any of the tested enzymes extracellularly. In natural isolates of Brevibacillus borstelensis LK 99 and Aneurinibacillus thermoaerophilus LK 102, only the ability to produce lipolytic enzymes was detected. The isolate Brevibacillus borstelensis Bz acts as a universal producer of all selected extremozymes. Enzyme activity was determined for selected producers. The bacterium Brevibacillus borstelensis Bz proved the ability to produce all six selected hydrolytic enzymes and has the highest activity of lipases, xylanases, cellulases and pectinases from the tested microorganisms. The highest proteolytic activity was measured in Thermomonas hydrothermalis DSM 14834 when cultured on skimmed milk powder.
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