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A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes
L P S Alves1, A T Almeida1, L M Cruz1
1Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
Summary
Quantifying poly-3-hydroxybutyrate (PHB) in bacteria is now faster. A new flow cytometry method using Nile red staining offers a rapid and accurate alternative to traditional techniques for PHB analysis.
Area of Science:
- Microbiology
- Biotechnology
- Analytical Chemistry
Background:
- Polyhydroxyalkanoates (PHAs), including poly-3-hydroxybutyrate (PHB), are biopolyesters with significant industrial applications.
- Traditional methods for PHB quantification, such as whole-cell methanolysis followed by gas chromatography (GC), are often laborious and time-consuming.
- Efficient and rapid quantification methods are crucial for optimizing PHA production in bacteria.
Purpose of the Study:
- To develop and validate a rapid flow cytometry-based method for quantifying poly-3-hydroxybutyrate (PHB) production.
- To compare the accuracy and efficiency of the new method against the conventional methanolysis-GC technique.
- To apply the optimized method for PHB quantification in plant-associated bacteria.
Main Methods:
- Optimization of a three-step protocol: cell permeabilization, Nile red staining, and flow cytometry analysis.
- Validation of the flow cytometry method against standard whole-cell methanolysis and GC analysis.
- Application of the method to quantify PHB in Herbaspirillum seropedicae and Azospirillum brasilense, including epiphytic bacteria from rice roots.
Main Results:
- The developed flow cytometry method for PHB quantification can be completed in under 5 minutes.
- A high correlation coefficient (R2=0.99) was achieved when comparing flow cytometry results to the standard methanolysis-GC method.
- The method demonstrated successful application in quantifying PHB in epiphytic bacteria isolated from rice roots.
Conclusions:
- A rapid, accurate, and efficient flow cytometry-based method for PHB quantification has been established.
- This method significantly reduces the time and labor associated with PHB analysis compared to conventional techniques.
- The validated method is suitable for quantifying PHB in various bacterial species, including important plant-associated diazotrophs and epiphytes.
