Microscopic examination in vivo and in vitro of natural and cross-linked polyunsaturated mclPHA

Pseudomonas aeruginosa 42A2 produces a polyunsaturated polyhydroxyalkanoates (PHA-L) when grown on linseed oil as a substrate. Its high unsaturation content (36.5%) provides highly reactive PHA-L, generating a cross-linked biopolymer after ultraviolet (UV) irradiation. Both PHAs (PHA-L and uvPHA-L) were characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, gel permeation chromatography, gas chromatography–mass spectrometry and differential scanning calorimetry–thermogravimetric analysis. The structural analysis of the new polymer revealed a dramatic decrease in unsaturated monomer content (8.5%), due to the complete disappearance of the polyunsaturated monomers (C_12:2, C_14:2, and C_14:3). The cross-linking reaction was also confirmed by atomic force microscopy (AFM) and transmission electron microscopy. AFM showed morphological changes in bacteria cells with and without PHA granules. The microscope techniques provided us with micrographs of the native and cross-linked polymers, showing the formation of a reticular structure as the consequence of the cross-linking reaction.