Monitoring the in situ crystallization of native biopolyester granules in Ralstonia eutropha via infrared spectroscopy

Poly(3-hydroxybutyrate) (PHB), a representative polyhydroxyalkanoate (PHA), is a naturally occurring biopolyester stored as tiny, intracellular granules in microbial cells. In vivo, native PHB granules are amorphous, stabilized by a monolayer membrane and intra-granule water. When subjected to varying environmental conditions, the native granules may become partially crystalline. The in situ crystallinity of native PHB granules in Ralstonia eutropha cells suspended in aqueous solution was monitored with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). No sample preparation was required for measurement. A major measurement error could be caused by the evaporation of water. Therefore, the infrared absorption spectra should be taken after the initial settlement of cells, but before excessive dehydration. Background interference caused by water and non-PHB biomass was constant throughout the time course of measurement, regardless of granule crystallinity. The wavenumber 1184 cm⁻¹ was found to be most sensitive to the in situ crystallinity of native PHB granules.