The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator

Alexander Kettner; Carola Griehl

doi:10.1016/j.bbrep.2020.100819

The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator

Biochem Biophys Rep. 2020 Sep 30:24:100819. doi: 10.1016/j.bbrep.2020.100819. eCollection 2020 Dec.

Authors

Alexander Kettner¹, Carola Griehl¹

Affiliation

¹ Anhalt University of Applied Sciences, Department of Applied Biosciences and Process Engineering, Bernburger Strasse 55, 06366, Koethen, Germany.

Abstract

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490-520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.

Keywords: Bioimaging; Cupriavidus necator; Fluorescence; LipidGreen2; Poly(3-hydroxybutyrate); Polyhydroxyalkanoates.