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Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (oponent) ; Sedláček, Petr (vedoucí práce)
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.
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Enkapsulace půdních bakterií v hydrogelových nosičích
Orišková, Sofia ; Smilek, Jiří (oponent) ; Sedláček, Petr (vedoucí práce)
Náplňou bakalárskej práce bolo vypracovať literárnu rešerš na tému inokulácie pôdnych baktérií, zhrnutím enkapsulačných techník a nosičových systémov vhodných pre poľnohospodárske účely. Na základe týchto údajov bol navrhnutý modelový systém enkapsulácie, ktorého optimalizáciou sa zaoberá experimentálna časť tejto práce. Enkapsulácia umožňuje postupné uvoľňovanie pôdnych baktérií, chráni a stabilizuje ich, čím zaručuje dlhšiu trvanlivosť. Pre inokuláciu bola vybraná dusík fixujúca baktéria Cupriavidus necator H16, ktorá bola enkapsulovaná do alginátu, s ktorým tvorila agregátne mikrokapsule. Enkapsulácia prebiehala na základe iónovej gelácie, kedy alginát sodný reagoval so sieťovacím činidlom, chloridom vápenatým, za tvorby kapsúl. Sledoval sa vplyv rôznych faktorov na kvalitu kapsúl. Zistilo sa, že molekulárna hmotnosť alginátu výrazne ovplyvňuje úspešnosť sieťovania. Alginát s vyššou strednou molekulárnou hmotnosťou sa vyhodnotil ako vhodnejší pre daný systém. Ako najvhodnejšie disperzné prostredie sa určila destilovaná voda, poprípade tlmivý roztok TRIS-HCl, ten bol však nevhodný v prípade potreby získania suchého produktu lyofilizáciou. Ďalej sa skúmala viabilita enkapsulovaných buniek. Zistilo sa, že bunky enkapsuláciu prežili a postupne sa z kapsúl uvoľňujú. Bolo overené, že pri vhodne zvolených podmienkach a zložení systému je možné baktériu C. necator H16 enkapsulovať do alginátových nosičov. Preukázala sa aj viabilita baktérií po enkapsulácii.
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Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (oponent) ; Sedláček, Petr (vedoucí práce)
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.
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Enkapsulace půdních bakterií v hydrogelových nosičích
Orišková, Sofia ; Smilek, Jiří (oponent) ; Sedláček, Petr (vedoucí práce)
Náplňou bakalárskej práce bolo vypracovať literárnu rešerš na tému inokulácie pôdnych baktérií, zhrnutím enkapsulačných techník a nosičových systémov vhodných pre poľnohospodárske účely. Na základe týchto údajov bol navrhnutý modelový systém enkapsulácie, ktorého optimalizáciou sa zaoberá experimentálna časť tejto práce. Enkapsulácia umožňuje postupné uvoľňovanie pôdnych baktérií, chráni a stabilizuje ich, čím zaručuje dlhšiu trvanlivosť. Pre inokuláciu bola vybraná dusík fixujúca baktéria Cupriavidus necator H16, ktorá bola enkapsulovaná do alginátu, s ktorým tvorila agregátne mikrokapsule. Enkapsulácia prebiehala na základe iónovej gelácie, kedy alginát sodný reagoval so sieťovacím činidlom, chloridom vápenatým, za tvorby kapsúl. Sledoval sa vplyv rôznych faktorov na kvalitu kapsúl. Zistilo sa, že molekulárna hmotnosť alginátu výrazne ovplyvňuje úspešnosť sieťovania. Alginát s vyššou strednou molekulárnou hmotnosťou sa vyhodnotil ako vhodnejší pre daný systém. Ako najvhodnejšie disperzné prostredie sa určila destilovaná voda, poprípade tlmivý roztok TRIS-HCl, ten bol však nevhodný v prípade potreby získania suchého produktu lyofilizáciou. Ďalej sa skúmala viabilita enkapsulovaných buniek. Zistilo sa, že bunky enkapsuláciu prežili a postupne sa z kapsúl uvoľňujú. Bolo overené, že pri vhodne zvolených podmienkach a zložení systému je možné baktériu C. necator H16 enkapsulovať do alginátových nosičov. Preukázala sa aj viabilita baktérií po enkapsulácii.
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