Differential scanning calorimetric study of poly(3-hydroxyoctanoate) inclusions in bacterial cells

Medium chain length polyhydroxyalkanoates, MCL-PHAs, produced by bacteria as inclusion bodies or granules were analyzed in situ by differential scanning calorimetry (DSC) without isolation from the cells. The kinetic DSC study of PHA granules, which contained mostly 3-hydroxyoctanoate units (PHO), in Pseudomonas putida BM01 cells showed that the polymer within the granules existed in an amorphous state, but it crystallized after dehydration of the cells under freeze-drying condition (below -50 degrees C) followed by annealing at ambient temperature. In this manner, PHO within the cells readily crystallized to the maximum degree of crystallinity within 24 h at room temperature, which was much faster than for the same polymer isolated by solvent extraction. This observation suggests that the polymer within the cellular granules may be well organized. The DSC endothermic melting peak areas for the room-temperature annealed polymers within the cells were directly proportional to the amount of polymer in the cell, and the results from this type of quantitative analysis were essentially identical to those obtained by gas chromatographic and gravimetric analysis of the polymers. X-Ray diffraction analysis of the polymer in the freeze-dried, whole cells and of the isolated, fully crystallized polymer showed that the two types of PHO samples had similar crystal structures, but the polymer in the granules exhibited better side-chain packing and higher crystallinity.