Robust multi-resolution analysis of polyhydroxyalkanoate biopolymers synthesized in a wastewater bacterium

Polyhydroxyalkanoates are microbial carbon-storage biopolymers with potential to replace fossil fuel-derived plastics and other polymers, but comparative analysis of the methods for characterization of these biopolymers is lacking. Here, we analyzed polyhydroxyalkanoates in a wastewater-isolated Comamonas species using fluorescence microscopy and flow cytometry for visualization and semi-quantitative assessment, followed by single-cell Raman spectroscopy, gas chromatography-flame ionization detection, and liquid chromatography-mass spectrometry for compositional quantification. Results show that fluorescence microscopy and flow cytometry revealed consistently nutrient-dependent accumulation, with nitrogen-limited cultures exhibiting up to 7-fold higher intracellular fluorescence intensity than controls. Single-cell Raman spectroscopy identified, in addition to low-abundant glycogen-like polymer, coexistence of high-abundant poly(3-hydroxybutyrate) and poly(3-hydroxyvalerate). Gas chromatography-flame ionization detection determined that these short-chain-length polyhydroxyalkanoates constituted 28% of nitrogen-limited biomass. With a derivatization-free high-resolution liquid chromatography-mass spectrometry method, we confirmed minimal glucan, less than (<0.05%) and resolved composition of polyhydroxyalkanoates as 97.8% 3-hydroxybutyrate, 1.7% 3-hydroxyvalerate, and 0.5% medium-chain-length monomers as 3-hydroxydecanoate and 3-hydroxytetradecanoate, demonstrating a sensitive approach with detection limits below 0.05 µg mL− 1 for biopolymer quantification.