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Utilization of vibrational spectroscopy for in-situ qualitative and quantitative analysis of polyhydroxyalkanoates in microbial biomass
Kevélyová, Barbora ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis was to evaluate the applicability of selected methods of vibrational spectroscopy for qualitative and quantitative analysis of biomass, which contains polyhydroxyalkanoates. Fourier-transform infrared spectroscopy, that uses attenuated total reflection technique (ATR-FTIR), was chosen for this experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) has been studied in Cupriavidus malaysiensis, Thermomonas hydrothermalis and Aneurinibacillus thermoaerophilus sp. H1. Data were obtained by evaluating the infrared spectra, which were then compared with the results of performed gas chromatography. Visual changes in the spectra were also observed along with crystallinity. No correlation was detected between the data obtained by gas chromatography and by the method ATR-FTIR in quantitative analysis. Thus, this method is not suitable for determining the content of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in biomass in the set experiment and the bacteria used. On the other hand, the method was successfully used for in-situ qualitative analysis of copolymer in the samples of Aneurinibacillus thermoaerophilus sp. H1. The degree of data correlation was relatively high in the given experiment. With these findings, ATR-FTIR is a suitable analytical method for determining the monomer composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in this strain. Comparison with the PHA producer Cupriavidus malaysiensis showed that this method is not universal for all types of microorganisms observed in this thesis.
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Preparation and application particles from copolymer of P(3HB-co-4HB)
Ostanina, Kseniia ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with preparation of particles from poly(3-hydroxybutyráte-co-4-hydroxybutyráte). Copolymer was dissolved in acetone and then the solution had been dosed into the anti-solvents. Acetone and anti-solvent are perfectly miscible. Theoretical part focuses on the properties of the copolymer, its production and potential use in medical applications. In the experimental part the copolymer was dripped into selected anti-solvents by syringe and pipette above and below the surface of anti-solvent. The following anti-solvent were chosen: water, methanol, dimethylsulfoxid (DMSO), 50 % acetone-water, 75% acetone-water, 50 % methanol-water, 75 % methanol-water mixtures. There were three copolymers used with different compositions of 4HB units (63 %, 87 % a 33 %). Copolymer composition was analyzed by gas chromatography. Copolymers were prepared by bacterial strain Aneurinibacillus sp. H1. Due to the results particles of size approximately 100-500 nm can be prepared by methods described in this work. The produced particles can be successfully used in medical applications.
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Rheological properties and biodegradation of Poly (3-hydroxybutyrate-co-4-hydroxybutyrate)
Černeková, Nicole ; Obruča, Stanislav (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis deals with the characterization of rheological properties and biodegradation of a poly(3-hydroxy-co-4-hydroxybutyrate), a copolymer produced by a strain of Cupriavidus malaysiensis. The theoretical part focuses on the rheology and degradation of polymers in general. It also contains the basic characteristics of polyhydroxyalkanoates and deals in more details with the description of the studied copolymer, its properties, synthesis, decomposition and possibilities of its applications. In the experimental part, the given copolymer was investigated in its native form and also in the form of copolymer mixtures filaments, which were enriched with additives (stabilizers and plasticizers). In terms of rheology, the samples showed pseudoplastic behaviour, which was slightly limited by the plasticization of the material. The highest thermal stability observed as a function of changes in complex viscosity over time was observed in a sample of a copolymer mixture containing stabilizers and plasticizer Citrofol BII. The plasticization also caused significant changes in thermal properties, especially crystallinity, which decreased compared to the native copolymer. In vitro degradation studies of samples in the form of films prepared by dissolving copolymer mixtures in chloroform were performed in phosphate buffer with lipase, in simulated body fluid and in synthetic gastric juice. The course of the process itself was characterized by the method of determining the molecular weight (SEC-MALLS) and the weight loss over time (gravimetrically). The results showed that the analysed samples are subject to degradation in all used environments. The most considerable molecular weight loss after 105 days (76 %) was observed in the sample containing stabilizers and plasticizer Citrofol BII in the environment of synthetic gastric juice. The most extensive gravimetric weight loss was attributed to the sample with stabilizers and without plasticizers, in the environment of phosphate buffer with lipase, specifically by 79%.
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Study of the release of active substances from PHA-based particles
Čech, Jan ; Mikulíková, Renata (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the study of the release kinetics of caffeic acid and acetylsalicylic acid from polylactic acid, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate)-based microparticles. In the theoretical part, the mechanical and thermal properties of polyhydroxyalkanoates, their biological synthesis by microorganisms, their industrial extraction from biomass and applications are described. Furthermore, the properties of PHA microparticles, their use as carriers, the most common microparticle preparations and the release method of the loaded drug are described. In the experimental part, PLA, P(3HB) and P(3HB-co-4HB) based microparticles prepared by the solvent evaporation emulsion method were characterized by dynamic light scattering. Subsequently, caffeic acid or acetylsalicylic acid was incorporated into these microparticles, and the release kinetics of the active ingredient was observed spectrophotometrically for one week. Once measurement of the releasing kinetics ended, microparticles were again measured by DLS and compared with the characterized microparticles before releasing kinetics. Finally, the encapsulation efficiency and microparticle recovery were calculated.
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Preparation and application particles from copolymer of P(3HB-co-4HB)
Ostanina, Kseniia ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with preparation of particles from poly(3-hydroxybutyráte-co-4-hydroxybutyráte). Copolymer was dissolved in acetone and then the solution had been dosed into the anti-solvents. Acetone and anti-solvent are perfectly miscible. Theoretical part focuses on the properties of the copolymer, its production and potential use in medical applications. In the experimental part the copolymer was dripped into selected anti-solvents by syringe and pipette above and below the surface of anti-solvent. The following anti-solvent were chosen: water, methanol, dimethylsulfoxid (DMSO), 50 % acetone-water, 75% acetone-water, 50 % methanol-water, 75 % methanol-water mixtures. There were three copolymers used with different compositions of 4HB units (63 %, 87 % a 33 %). Copolymer composition was analyzed by gas chromatography. Copolymers were prepared by bacterial strain Aneurinibacillus sp. H1. Due to the results particles of size approximately 100-500 nm can be prepared by methods described in this work. The produced particles can be successfully used in medical applications.
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Utilization of vibrational spectroscopy for in-situ qualitative and quantitative analysis of polyhydroxyalkanoates in microbial biomass
Kevélyová, Barbora ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
The aim of this bachelor thesis was to evaluate the applicability of selected methods of vibrational spectroscopy for qualitative and quantitative analysis of biomass, which contains polyhydroxyalkanoates. Fourier-transform infrared spectroscopy, that uses attenuated total reflection technique (ATR-FTIR), was chosen for this experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) has been studied in Cupriavidus malaysiensis, Thermomonas hydrothermalis and Aneurinibacillus thermoaerophilus sp. H1. Data were obtained by evaluating the infrared spectra, which were then compared with the results of performed gas chromatography. Visual changes in the spectra were also observed along with crystallinity. No correlation was detected between the data obtained by gas chromatography and by the method ATR-FTIR in quantitative analysis. Thus, this method is not suitable for determining the content of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in biomass in the set experiment and the bacteria used. On the other hand, the method was successfully used for in-situ qualitative analysis of copolymer in the samples of Aneurinibacillus thermoaerophilus sp. H1. The degree of data correlation was relatively high in the given experiment. With these findings, ATR-FTIR is a suitable analytical method for determining the monomer composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in this strain. Comparison with the PHA producer Cupriavidus malaysiensis showed that this method is not universal for all types of microorganisms observed in this thesis.
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Rheological properties and biodegradation of Poly (3-hydroxybutyrate-co-4-hydroxybutyrate)
Černeková, Nicole ; Obruča, Stanislav (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis deals with the characterization of rheological properties and biodegradation of a poly(3-hydroxy-co-4-hydroxybutyrate), a copolymer produced by a strain of Cupriavidus malaysiensis. The theoretical part focuses on the rheology and degradation of polymers in general. It also contains the basic characteristics of polyhydroxyalkanoates and deals in more details with the description of the studied copolymer, its properties, synthesis, decomposition and possibilities of its applications. In the experimental part, the given copolymer was investigated in its native form and also in the form of copolymer mixtures filaments, which were enriched with additives (stabilizers and plasticizers). In terms of rheology, the samples showed pseudoplastic behaviour, which was slightly limited by the plasticization of the material. The highest thermal stability observed as a function of changes in complex viscosity over time was observed in a sample of a copolymer mixture containing stabilizers and plasticizer Citrofol BII. The plasticization also caused significant changes in thermal properties, especially crystallinity, which decreased compared to the native copolymer. In vitro degradation studies of samples in the form of films prepared by dissolving copolymer mixtures in chloroform were performed in phosphate buffer with lipase, in simulated body fluid and in synthetic gastric juice. The course of the process itself was characterized by the method of determining the molecular weight (SEC-MALLS) and the weight loss over time (gravimetrically). The results showed that the analysed samples are subject to degradation in all used environments. The most considerable molecular weight loss after 105 days (76 %) was observed in the sample containing stabilizers and plasticizer Citrofol BII in the environment of synthetic gastric juice. The most extensive gravimetric weight loss was attributed to the sample with stabilizers and without plasticizers, in the environment of phosphate buffer with lipase, specifically by 79%.
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