Fermentation Monitoring of Yeast Using Size Exclusion-ligand Exchange (Na-type) Column

Significance of the Topic

Accurate quantification of saccharides and ethanol in microbial fermentation is vital for optimizing yields, ensuring consistent product quality, and guiding process control in industries such as biofuels and alcoholic beverages.

Objectives and Study Overview

This study establishes a retention index for twenty saccharides and sugar alcohols using a Shim-pack SUR-Na column on a Nexera™ lite HPLC system with water as the sole mobile phase. It further demonstrates calibration curves for key analytes and applies the method to real-time monitoring of yeast fermentation in molasses.

Methodology and Instrumentation

A Shimadzu Nexera lite isocratic HPLC system equipped with a RID-20A refractive index detector was configured with:

• Shim-pack SUR-Na analytical column (250×7.8 mm, 8 µm) and guard column (50×7.8 mm, 8 µm)
• Mobile phase: ultrapure water
• Column temperature: 80 °C
• Flow rates: 0.6 mL/min (single column), 0.5 mL/min (two columns in series)
• Injection volumes: 20 µL for retention index, 2 µL for standards and samples

The separation combines size exclusion and Na-type ligand exchange to resolve oligosaccharides, monosaccharides, and sugar alcohols without additional reagents.

Main Results and Discussion

The retention index for twenty analytes ranged from 7.7 min (raffinose) to 15.6 min (ethanol) on a single column, extending to 16.4–33.8 min with two columns in series. Calibration curves for sucrose, glucose, fructose, and ethanol exhibited excellent linearity (R² ≥ 0.999999). In fermentation samples, sucrose consumption correlated with rising glucose and fructose levels, which were subsequently converted to ethanol. Concentration–time profiles clearly traced the progression of saccharide hydrolysis and ethanol production.

Benefits and Practical Applications

• Uses only water as mobile phase, simplifying preparation and reducing cost
• High-resolution separation of a wide range of saccharides and sugar alcohols
• Rapid establishment of retention indices for routine QA/QC
• Real-time monitoring of fermentation dynamics for process optimization

Future Trends and Potential Applications

Integration of this robust methodology into process analytical technology (PAT) frameworks could enable inline fermentation monitoring. Future developments may include coupling with mass spectrometry for enhanced sensitivity, miniaturized HPLC platforms for on-site analysis, and extension to other bioprocess-derived compounds such as organic acids and peptides.

Conclusion

The combination of size exclusion and sodium-type ligand exchange on the Shim-pack SUR-Na column, together with a simple water mobile phase on a Nexera™ lite system, provides a powerful, reproducible approach for saccharide separation and fermentation monitoring. Its ease of use and broad applicability make it a valuable tool for food, beverage, and bioenergy sectors.