Chlorinated-solvent-free gas chromatographic analysis of biomass containing polyhydroxyalkanoates

An extraction and derivatization method was developed for more environmentally friendly routine quantification of polyhydroxyalkanoates (PHAs) in activated sludge biomass by gas chromatography (GC). This method can be further applied to assess relative changes in biomass carbohydrate levels relating to, for example, glycogen or extracellular polysaccharides. Further, co-extracted principal membrane fatty acids are indicative of relative changes in active biomass. The protocol is attractive for routine assessment because it does not require any chlorinated solvents, which are still almost exclusively used today for PHA analysis by GC. Acidic alcoholysis of dried microbial biomass using 3:1 butanol to concentrated (37%) hydrochloric acid at 100 °C for 8 h will hydrolyse and derivatize microbial storage products and membrane lipids. Esters of the hydroxyalkanoates, carbohydrates converted to levulinic acid, and long chain microbial fatty acids are reliably extracted into hexane for gas chromatographic analysis and quantification. Calibration can be achieved with benzoic, 2-hydroxyvaleric, or 2-hydroxycaproic acids as the method reference standards.