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Involvement of vibrational spectroscopy in the in-situ analysis of PHA in bacterial biomass.
Kevélyová, Barbora ; Samek, Ota (referee) ; Sedláček, Petr (advisor)
The aim of this thesis was the study of polyhydroxyalkanoates in bacterial biomass using the ATR-FTIR method. Furthermore, the PCA method was chosen to evaluate the measured data and observe correlations or differences among the spectra of various biomass samples. The bacterial producents were Aneurinibacillus thermoaerophilus H1, AH30 and AFn2, Cupriavidus malaysiensis DSM 25816, DSM 19416 and DSM 19379 and three strains of Schlegelella thermodepolymerans M 15344, DSM 15264 and LMG 21645. Various heteropolymers with different monomer content and total PHA content were investigated. The monomers in the samples were 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxyvalerate, 5-hydroxyvalerate and 4-hydroxyhexanoate. As the reference method for accurate quantitative and qualitative determination of polyhydroxyalkanoates in the samples, gas chromatography was performed. The spectra obtained by the FTIR method were compared and significant peaks related to the qualitative properties of PHA, namely monomer composition and crystallinity, were searched for. The PCA statistical method was successfully applied to the collected data from FTIR, with the help of which the qualitative differences between the samples were monitored. The most significant differences were observed in the presence of monomers 4-hydroxybutyrate and 4-hydroxyhexanoate, and they were related to the crystallinity of the samples. The analysis did not show potential for semi-quantitative determination of PHA in biomass. Differences between microorganisms were not observed. The procedure for evaluating FTIR spectra using PCA could in the future be used in the study of biomass containing various monomeric units, especially hydroxyvalerates in case of their higher content within the sample.
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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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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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