Inkorporace mikrobiálních buněk do hydrogelových nosičů
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Diplomová práca vychádza z moderného agrochemického konceptu využitia pôdnych baktérií podporujúcich rast rastlín ako ekologickej alternatívy konvenčného hnojenia. Inkorporácia bakteriálnych buniek do hydrogélových nosičov je už preštudovaný proces, no pre svoje nevýhody zatiaľ nenachádza širšie uplatnenie v poľnohospodárstve. Táto práca ponúka úplne nový koncept enkapsulácie baktérií geláciou priamo z kultúry. Tohto efektu sa dosiahne sieťovaním bakteriálneho alginátu, ktorý je produkovaný modelovým mikroorganizmom Azotobacterom vinelandii. Šlo o pilotné experimenty, kedy boli optimalizované kultivačné, ako aj gelačné podmienky. Gravimetricky bola stanovená koncentrácia alginátu, ktorého parametre boli charakterizované použitím vybraných analytických metód – infračervenej spektroskopie pre monitoring kľúčových štruktúrnych parametrov (monomérnej kompozície a miery acetylácie), dynamického rozptylu svetla na charakteristiku distribúcie veľkosti či AF4-MALS-dRI na stanovenie molekulovej hmotnosti. Azotobacter vinelandii je tiež producentom PHB, ktorého tvorba bola sledovaná pomocou plynovej chromatografie a infračervenej spektroskopie. Druhá časť práce je zameraná na optimalizáciu tvorby gélov sieťovaním bakteriálnej kultúry s vyprodukovaným alginátom pomocou CaCl2, kedy ku kvalitatívnemu popisu vzniknutej gélovej kompozície slúžili reometrické experimenty. Bola dokázaná možnosť gelácie už po 24 h od naočkovania kultúry. Maximálna produkcia alginátu (1,9 ± 0,3) g/l bola dosiahnutá v štvrtý deň od naočkovania. Bolo tiež zistené, že prídavok 5 g/l uhličitanu vápenatého podporí tvorbu alginátu, no vyšší prídavok CaCO3 (30 g/l) negatívne vplýva na molekulovú hmotnosť alginátu, preto sa neodporúča. Pomocou FTIR aj GC bola potvrdená tvorba PHB, s maximálnym výťažkom (23 ± 3) % CDW. Reologické testy potvrdili, že produktom sieťovania kultúry je gél. V čase 0 min bol sledovaný vplyv koncentrácie sieťovacieho činidla a bolo zistené, že s vyššou koncentráciou vápenatých iónov je sprevádzaná tvorba hustejšej sieti v štruktúre a vyššia rigidita gélu. Pri najvyššej skúmanej koncentrácii sieťovadla CaCl2 bola hranica lineárnej viskoelastickej oblasti určená na (5,0 ± 0,7) %. Na záver bola fotografiami potvrdená inkorporácia bakteriálnych buniek do gélu využitím lipofilného fluorescenčného farbiva BODIPY 439/503.
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.
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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Citation
ORIŠKOVÁ, S. Inkorporace mikrobiálních buněk do hydrogelových nosičů [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2020.
Document type
Document version
Date of access to the full text
Language of document
sk
Study field
Spotřební chemie
Comittee
prof. Ing. Miloslav Pekař, CSc. (předseda)
prof. Ing. Michal Veselý, CSc. (místopředseda)
prof. Ing. Michal Čeppan, Ph.D. (člen)
doc. Ing. Zdenka Kozáková, Ph.D. (člen)
doc. Mgr. Aleš Mráček, Ph.D. (člen)
Date of acceptance
2020-09-01
Defence
Obhajoba proběhla podle následujícího schématu: prezentace studentky-vyjádření vedoucí/ho-oponentský posudek-reakce na posudek-diskuse s komisí. Studentka přednesla výborný výtah výsledků své diplomové práce, řádně zodpověděla všechny dotazy oponentské i členů komise, pohotově reagovala na připomínky. V diskusi tak studentka prokázala výbornou schopnost orientace v teoretických i praktických základech problematiky diplomové práce. Komise zhodnotila její diplomovou práci celkově jako výbornou.
Pekař: O jaký typ bakterie, které jste používala, se jedná?
Veselý: Nepoškodí lyofilizace bakterie?
Čeppan: Čím je způsobena velká nejistota měření?
Kozáková: Jakým způsobem by se gel v praxi aplikoval?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení