Combined Determination of Poly-β-Hydroxyalkanoic and Cellular Fatty Acids in Starved Marine Bacteria and Sewage Sludge by Gas Chromatography with Flame Ionization or Mass Spectrometry Detection

Abstract

Extraction of lipids from bacterial cells or sewage sludge samples followed by simple and rapid extraction procedures and room temperature esterification with pentafluorobenzylbromide allowed combined determinations of poly-beta-hydroxyalkanoate constituents and fatty acids. Capillary gas chromatography and flame ionization or mass spectrometric detection was used. Flame ionization permitted determination with a coefficient of variation ranging from 10 to 27% at the picomolar level, whereas quantitative chemical ionization mass spectrometry afforded sensitivities for poly-beta-hydroxyalkanoate constituuents in the attomolar range. The latter technique suggests the possibility of measuring such components in bacterial assemblies with as few as 10 cells. With the described technique using flame ionization detection, it was possible to study the rapid formation of poly-beta-hydroxyalkanoate during feeding of a starved marine bacterium isolate with a complex medium or glucose and correlate the findings to changes in cell volumes. Mass spectrometric detection of short beta-hydroxy acids in activated sewage sludge revealed the presence of 3-hydroxybutyric, 3-hydroxyhexanoic, and 3-hydroxyoctanoic acids in the relative proportions of 56, 5 and 39%, respectively. No odd-chain beta-hydroxy acids were found.