Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red...

Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence microscopy qualitatively. Our second approach enables the conversion of PHB to volatile hydroxycarboxylic acid methyl esters by acidic methanolysis and their quantification by gas chromatography. Through this method, it is possible to obtain an absolute quantification of PHB, e.g., per cell dry weight.