The determination of carbohydrates, alcohols, and glycols in fermentation broths

Significance of the Topic

Fermentation broths are key to producing biotherapeutics and biofuels. Accurate profiling of carbon sources (sugars) and metabolic by-products (alcohols, glycols) is essential because these components directly influence yield and process efficiency. Many fermentation constituents lack chromophores and require specific detection methods.

Objectives and Study Overview

This application note compares two high-performance anion-exchange chromatography methods with pulsed amperometric detection (HPAE-PAD) for simultaneous analysis of simple sugars, sugar alcohols (alditols), alcohols, and glycols in yeast (Saccharomyces cerevisiae) and bacterial (E. coli) fermentation broths. Key performance metrics include selectivity, detection limits, linearity, precision, and stability.

Methodology and Instrumentation

• Instrumentation: Thermo Scientific Dionex DX-500 BioLC system (GP40 pump, ED40 detector, LC-oven, S3500 autosampler) with PeakNet workstation.
• Columns: CarboPac MA1 and PA1 analytical and guard columns (polystyrene/divinylbenzene resins functionalized with quaternary ammonium groups).
• Eluents: Sodium hydroxide solutions (16–480 mM) prepared from 50 % NaOH, blanketed with helium, and online-degassed to remove CO₂ and O₂.
• PAD Settings: Triple-potential waveform on gold electrode (0.05 V to +0.75 V to –0.15 V), 30 °C, 10 µL injection.
• Sample Preparation: Centrifugation and dilution (10–100×) of fermentation supernatants; spiking of standards into heat-treated broth for method evaluation.

Main Results and Discussion

• Column Selectivity: MA1 separates alcohols, glycols, alditols, mono-, di-, and trisaccharides (run times up to 60 min); PA1 offers faster separation and improved di/trisaccharide elution (run times 30–40 min).
• Detection Limits (MA1, 10 µL): glycerol 0.4 µg/L; 2,3-butanediol 1 ng; ethanol 300 ng; methanol 7 µg; maltose 900 ng.
• Linearity: 3–4 orders of magnitude (0.04–10 000 ng) with r²>0.998 for most analytes; glycerol, erythritol, galactitol required second-order fit at highest range.
• Precision & Stability: Peak area RSD 2–7 %; retention time RSD 0.2–0.4 % over 48 h.
• Application to Broths: Yeast cultures showed rapid glucose consumption, glycerol and ethanol production, and differential utilization of disaccharides and alditols. E. coli broth contained minimal carbohydrates and displayed distinct metabolic profiles.

Benefits and Practical Applications

• Derivatization-free analysis of nonchromophoric fermentation components.
• Broad dynamic range reduces need for repeated dilutions.
• High sensitivity and selectivity support process monitoring, QA/QC, and optimization in biomanufacturing and biofuel production.

Future Trends and Potential Applications

• Adoption of four-potential PAD waveforms and PTFE electrodes to further lower detection limits.
• Integration into online, real-time process control systems for fermentation monitoring.
• Extension to additional nonstandard sugars and organic anions to broaden analytical scope.

Conclusion

HPAE-PAD using Dionex CarboPac MA1 and PA1 columns on the DX-500 BioLC platform delivers sensitive, selective, and robust simultaneous determination of carbohydrates, alcohols, and glycols in complex fermentation broths. The methods meet stringent detection, linearity, precision, and stability requirements and facilitate efficient process development and quality control.

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