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. 2018 Mar 5;8(5):e2748.
doi: 10.21769/BioProtoc.2748.

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

Janina R Juengert  1 , Stephanie Bresan  1 , Dieter Jendrossek  1
Affiliations

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

Janina R Juengert et al. Bio Protoc. .

Abstract

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.

Keywords: Acidic methanolysis; Gas chromatography; Nile red; Polyhydroxybutyrate (PHB); Ralstonia eutropha.

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Figures

Figure 1.
Figure 1.. Biosynthesis of PHB
Figure 2.
Figure 2.. Acidic methanolysis of PHA
Figure 3.
Figure 3.. Flow chart the Nile red staining
Figure 4.
Figure 4.. Preparation of an agarose pad for microscopy
Figure 5.
Figure 5.. Flow chart of the determination of PHB content using gas chromatography
Figure 6.
Figure 6.. Images of the acidic methanolysis steps of PHB.
Weighted cells (A), complete methanolysis sample before oil bath (B), sample after oil bath (C) and phase-separated sample with the bottom organic phase (white arrow, D).
Figure 7.
Figure 7.. Fluorescence micrographs of cells with and without PHB.
From left to right: bright field, Nile red, merged channels of bright field and Nile red. Upper panel: cells with PHB granules, Lower panel: cells without PHB granules.
Figure 8.
Figure 8.. Screenshot of a chromatogram and results tables (A) and representative chromatograms for 3HVME and 3HBME (B)
Figure 9.
Figure 9.. Standard curve for the determination of PHB weight.
One line connects the data points, the second line is the trend line used to generate the equation of the line.
Figure 10.
Figure 10.. Example of the growth (A) and PHB production (B) of R. eutropha H16 on NB-medium with 0.2% Na-gluconate (complex medium) or on mineral salts medium with 2% fructose
See this image and copyright information in PMC

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