Staining and quantification of poly-3-hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry

Abstract

Background: Poly [(R)-3-hydroxybutyric acid] (PHB) is a prokaryote storage material for carbon and energy that accumulates in cells under unbalanced growth conditions. Because this class of biopolymers has plastic-like properties, it has attracted considerable interest for biomedical applications and as a biodegradable commodity plastic. Current flow cytometric techniques to quantify intracellular PHB are based on Nile red. Here, an improved cytometric technique for cellular PHB quantification utilizing BODIPY 493/503 staining was developed. This technique was then automated using an automated flow cytometry system. Materials: Using flow cytometry, the fluorescence of Saccharomyces cerevisiae and Cupriavidus necator with varying PHB content after staining with BODIPY 493/503 and Nile red was compared, and automated staining techniques were developed for both cultures. Results: BODIPY 493/503 staining had less background staining, higher sensitivity and specificity to PHB, and higher saturation values than did Nile red staining. The developed automated staining procedure was capable of analyzing the PHB content of a bioreactor sample every 25 min and measured the average PHB content with accuracy comparable to offline GC analysis. Conclusion: BODIPY 493/503 produced an overall better staining for PHB than did Nile red. When combined with the automated system, this technique provides a new method for the online monitoring and control of bioreactors.