Adaptation to adverse osmotic conditions as a tool for evolutionary enginnering of bacteria

Bakalárska práca sa zaoberá využitím osmotického stresu ako nástroja evolučného inžinierstva PHA produkujúcich bakteriálnych kmeňov. Cieľom práce je posúdiť využiteľnosť adaptácie mikroorganizmov na hypoosmotické prostredie ako inžinierskeho nástroja na ovplyvnenie efektivity biosyntézy PHA. Teoretická časť práce sa zaoberá princípom evolučného inžinierstva, metódami tejto stratégie a vplyvom fyzikálnych faktorov na mikroorganizmus. V rámci experimentálnej časti bol realizovaný experiment adaptívnej laboratórnej evolúcie s využitím bakteriálneho kmeňa Halomonas halophila CCM 3662. Ako stresový faktor počas sériovej kultivácie bol aplikovaný osmotický tlak v podobe zníženej koncentrácie soli v produkčnom médiu. Za účelom vyvinutia PHA produkujúcich adaptovaných mutantných kmeňov boli jednotlivé pasáže analyzované pomocou metód spektrofotometrie, gravimetrie a GC-FID. Na základe výsledkov dlhodobej kultivácie bol zistený adaptačný potenciál kmeňa HH35, kultivovaného pri 35 g/l NaCl, u ktorého obsah biomasy a koncentrácia PHB vykazovali najvyššie hodnoty. Z jednotlivých generácií kmeňa HH35 bola pre podrobnejšiu analýzu vybraná 15. pasáž a 30. pasáž, ktoré spolu s kontrolným divokým kmeňom H. halophila, boli podrobené hyperosmotickému a hypoosmotickému šoku. Pomocou metód FC a TGA bola sledovaná stresová odpoveď bakteriálnych kmeňov, v snahe zistenia viability buniek či potenciálnych morfologických zmien. Vyizolované polyméry boli charakterizované FTIR analýzou.This bachelor thesis deals with the application of osmotic stress as a tool for evolutionary engineering of PHA producing bacterial strains. The aim of this thesis is to evaluate a bacterial adaptation to hypoosmotic environment, as an engineering tool in order to increase the production of PHA. The theoretical part focuses on the evolutionary engineering principle, methods of the strategy and the effect of physical factors on microorganism. The aim of experimental part was to performed an adaptive evolutionary experiment with the bacterial strain Halomonas halophila CCM 3662. Reduced osmotic pressure was used as a stressing factor during the serial cultivation. In order to generate PHA producing mutant strains, each passage was characterized using spectrophotometric and gravimetric method and by GC-FID. It was found that after the long-term cultivation, the mutant strain HH35, cultivated in 35 g/l NaCl, was associated with the highest biomass and PHB concentration. The 15th and 30th passages, along with the wild type strain H. halophila were subjected to further cell-robustness analysis with the application of hyper- and hypoosmotic shock. The stress response, viability of cells and morphological changes were analyzed using FC and TGA methods. Isolated polymers were characterized using FTIR analysis.

Preparation of biodegradable films from PHA copolymers and evaluation of their application potential

Diplomová práca sa zaoberá prípravou biodegradabilných filmov na báze kopolymérov PHA a následným preštudovaním ich materiálových vlastností. Hlavným cieľom práce je posúdiť aplikačný potenciál PHA kopolymérnych filmov v biomedicínskom priemysle. Teoretická časť práce sa zaoberá charakterizáciou polyhydroxyalkanoátov a ich kopolymérov, z hľadiska fyzikálnych vlastností, syntézy, biodegradácie či PHA producentov. V literárnej rešerši sú popísané súčasné trendy v spracovaní a využití PHA v biomedicíne. V experimentálnej časti práce bola prevedená kultivácia bakteriálnych kmeňov Aneurinibacillus sp. H1 a Cupriavidus necator H16. Počas kultivácie Aneurinibacillus sp. H1 boli použité rôzne pomery substrátov glycerolu a 1,4-butándiolu, v snahe získať kopolyméry P(3HB-co-4HB) o rôznom monomérnom zložení. Pomocou extrakcie boli zo získanej biomasy vyizolované polymérne filmy, ktoré boli charakterizované pomocou metód GC-FID, FTIR a SEC-MALS. Následne boli vybraté kopolymérne filmy s obsahom 4HB 65,25 a 43,61 hm. % a homopolymérny PHB film, ktoré boli podrobené uvoľňovacím experimentom. Na uvoľňovacie experimenty boli použité liečivá ibuprofén a kyselina acetylsalicylová, u ktorých bolo sledované uvoľňovanie z filmu do fyziologického roztoku pomocou UV-VIS spektroskopie. Na základe získaných dát bol overený aplikačný potenciál PHA kopolymérnych filmov ako nosičového systému v biomedicínskom využití.This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified.

Preparation of biodegradable films from PHA copolymers and evaluation of their application potential

This diploma thesis deals with the production of biodegradable PHA copolymer fims and the subsequent study of their material properties. The main goal of this thesis is to assess the application potential of PHA copolymer films in the biomedical industry. The theoretical part of the thesis deals with the characterization of polyhydroxyalkanoates and their copolymers, in terms of physical properties, synthesis, biodegradation or PHA producers. The current research in the processing and use of PHA in biomedicine is described in the literature search. The cultivation of bacterial strains of Aneurinibacillus sp. H1 and Cupriavidus necator H16 was done in the experimental part. Different ratios of glycerol and 1,4-butanediol substrates were used during the cultivation of Aneurinibacillus sp. H1, to obtain copolymers of P(3HB-co-4HB) with a different monomer composition. Polymer films were isolated from the obtained biomass by the extraction method and were characterized by GC-FID, FTIR and SEC-MALS methods. Subsequently, copolymer films containing of 65,25 and 43,61 wt. % 4HB and homopolymer PHB film were selected for release experiments. The drugs, ibuprofen and acetylsalicylic acid, were used for release experiments, their release from the polymer film into the physiological solution was monitored by UV-VIS spectroscopy. Based on the obtained data, the application potential of PHA copolymer films as a carrier system in biomedical use was verified