Fast procedure for the analysis of poly(hydroxyalkanoates) in bacterial cells by off-line pyrolysis/gas-chromatography with flame ionization detector

• Pyrolysis of PHAs produces 2-alkenoic acids as diagnostic pyrolysis markers.
• 2-Alkenoic acid amount is proportional to the PHAs amount in bacterial biomass.
• Each 2-alkenoic acid is correlated to the specific monomers present in PHAs.
• Py/GC–FID analysis is a fast method for PHAs quantification and characterization.

Poly(hydroxyalkanoates) (PHAs) are polyesters formed by saturated short chain hydroxyacids, among which 3-hydroxybutanoic (HB) and 3-hydroxypentanoic (3-hydroxyvalerate, HV) are the most common monomers of homopolymers (e.g. poly(3-hydroxybutyrate), PHB) and copolymers (e.g. poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHB-HC). The most widely used approach for their determination is the polymer methanolysis followed by gas chromatography–mass spectrometry (GC–MS) analysis of the methylated monomers; this procedure generally requires the use of additional reagents (e.g. sulfuric acid) and is performed with harmful chlorinated solvents, such as chloroform. The development of fast routine solventless methods for the quantitative determination of PHAs and their monomeric composition is highly desirable to reduce sample pretreatment, speed up the analysis and decrease overall costs. It has been reported that under thermal treatment (e.g. pyrolysis, Py), PHAs are degraded in high yield (>40%, w/wPHA) into the corresponding 2-alkenoic acid (e.g. crotonic acid from PHB). This work aimed at investigating this reaction for direct analysis of PHAs in bacterial cells. The sample was directly subjected to pyrolysis and trapped pyrolysis products were analyzed by GC–FID. Off-line Py/GC–FID was first optimized on pure polymers with different monomer composition (PHB, PHB-HV, PHB-HC) and then applied to bacterial samples deriving from both mixed microbial cultures or selected strains, containing various types and amounts of PHAs. The Py/GC–FID method provided RSD <15% range, limit of detection of 100 μg (1% PHAs in biomass), and results comparable to that of methanolysis (R2 = 0.9855), but with minimal sample pretreatment.