Improved intracellular PHA determinations with novel spectrophotometric quantification methodologies based on Sudan...

Area of Science:

• Biotechnology
• Microbiology
• Analytical Chemistry

Background:

• Intracellular polyhydroxyalkanoates (PHAs) are typically quantified using Sudan black (SB) dye.
• Previous work established direct quantification of PHA via SB absorbance in wet cell samples.
• Optimization of SB staining conditions for PHA assays is crucial for accurate quantification.

Purpose of the Study:

• To optimize Sudan black (SB) staining conditions for polyhydroxyalkanoates (PHAs) quantification.
• To develop a novel, rapid, indirect method for determining intracellular PHA content.
• To compare the efficiency and precision of the new indirect method with existing direct methods.

Main Methods:

• Utilized hybrid response surface methodology and desirability-function for optimizing SB assay conditions.
• Developed an indirect quantification method measuring SB absorbance in the supernatant of stained cell samples.
• Employed multivariate and linear regression analysis to correlate supernatant spectra with PHA content.

Main Results:

• Optimized SB amount and assay conditions for direct PHA determination.
• The indirect method provides faster PHA content determination (23 min) compared to the direct method (42 min).
• High correlation (R² = 0.91) and good prediction (RSE = 1.81%) were achieved for the indirect method, demonstrating its precision.

Conclusions:

• The developed indirect methodology offers a precise, rapid, economical, and environmentally friendly approach for intracellular PHA content determination.
• This method is suitable for basic laboratory equipment and can be used for rapid monitoring of PHA accumulation.
• Combined with the optimized direct method, it provides a robust system for PHA analysis in microbial studies.