Poly-β-hydroxyalkanoate determination in bacteria from aquatic samples

A method for the determination of the poly-β-hydroxyalkanoate (PHA) content of planktonic bacteria (bacterioplankton) in fresh water is described. PHA measurements help determine the nutritional status of a microbial community. The method consists of three steps: on shore water sample filtration, solvent extraction of the filter and gas chromatography analysis. A known volume of water is filtered in the field through a glass fiber filter applying vacuum with a manual pump. In situ sample processing is a key factor because of PHA liability. The filters are frozen at the sampling site in liquid nitrogen in a portable container and transported to the laboratory where they are freeze-dried. Dry filters are then extracted in hot chloroform for 4 h by using a Soxhlet extractor. The solvent, containing the dissolved polymer, is evaporated, and the residue is digested with a mixture of methanol and sulfuric acid (methanolysis). The methyl esters of the carboxylic acids that correspond to the monomers forming the polymer are then analyzed by gas chromatography. This method was tested in the field and improves the PHA determination that is used in laboratory cultures for application to natural environments. The nutritional status of natural microbial planktonic communities can thus be estimated by the PHA determination technique described.