Možnosti využití vibrační spektroskopie při in-situ analýze PHA v mikrobiální biomase

Abstract

Cieľom tejto diplomovej práce bolo štúdium polyhydroxyalkanoátov v mikrobiálnej biomase pomocou metódy ATR-FTIR. Ďalej bola zvolená štatistická metóda PCA pre vyhodnotenie nameraných dát a pozorované korelácie či odlišnosti. Bakteriálny producenti boli Aneurinibacillus thermoaerophilus H1, AH30 a AFn2, Cupriavidus malaysiensis DSM 25816, DSM 19416 a DSM 19379 a tri kmene Schlegelella thermodepolymerans M 15344, DSM 15264 a LMG 21645. Skúmané boli rozličné heteropolyméry s rôznym obsahom monomérov a celkovým obsahom PHA. Monoméry vo vzorkách boli 3-hydroxybutyrát, 4-hydroxybutyrát, 3-hydroxyvalerát, 4-hydroxyvalerát, 5-hydroxyvalerát a 4-hydroxyhexanoát. Ako porovnávacia analýza pre presné kvantitatívne a kvalitatívne stanovenie polyhydroxyalkanoátov vo vzorkách bola prevedená plynová chromatografia. Spektrá získané metódou FTIR boli porovnávané a boli hľadané signifikantné píky, ktoré súvisia s kvalitatívnymi vlastnosťami PHA, konkrétne monomérnym zložením a stupňom kryštalinity. Na zozbierané dáta z FTIR bola úspešne aplikovaná štatistická metóda PCA, pomocou ktorej boli sledované najmä kvalitatívne rozdiely medzi vzorkami. Najdôležitejšie rozdiely boli pozorované v prípade prítomnosti monoméru 4-hydroxybutyrátu a 4-hydroxyhexanoátu, a týkali sa stupňa kryštalinity vzoriek. Analýza nepreukázala potenciál pri semi-kvantitatívnom stanovení PHA v biomase. Rozdiely medzi mikroorganizmami neboli pozorované. Postup vyhodnotenia FTIR spektier pomocou PCA by bolo vhodné do budúcna využiť pri štúdiu biomasy s obsahom rozličných monomérnych jednotiek, najmä hydroxyvalerátov v prípade ich vyššieho obsahu vo vzorke.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.