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

Poy(hydroxyalkanoates) (PHAs) are polyesters formed by saturated short chain hydroxyacids, amongwhich 3-hydroxybutanoic (HB) and 3-hydroxypentanoic (3-hydroxyvalerate, HV) are the mostcommon 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 theirdetermination is the polymer methanolysis followed by gas chromatography–mass spectrometry(GC–MS) analysis of the methylated monomers; this procedure generally requires the use of additionalreagents (e.g. sulfuric acid) and is performed with harmful chlorinated solvents, such as chloroform. Thedevelopment of fast routine solventless methods for the quantitative determination of PHAs and theirmonomeric composition is highly desirable to reduce sample pretreatment, speed up the analysis anddecrease overall costs. It has been reported that under thermal treatment (e.g. pyrolysis, Py), PHAs aredegraded in high yield (>40%, w/wPHA) into the corresponding 2-alkenoic acid (e.g. crotonic acid fromPHB). This work aimed at investigating this reaction for direct analysis of PHAs in bacterial cells. Thesample 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 culturesor selected strains, containing various types and amounts of PHAs. The Py/GC–FID method providedRSD <15% range, limit of detection of 100 g (1% PHAs in biomass), and results comparable to that ofmethanolysis (R2= 0.9855), but with minimal sample pretreatment.