Determination of Inorganic Anions and Organic Acids in Fermentation Broths

Importance of topic

Monitoring organic acids and inorganic anions in fermentation broths is essential for optimizing bioprocess yields and ensuring product quality. Fermentation by-products such as lactate, acetate, and formate can inhibit microbial growth and reduce target product formation. Simultaneous quantification of these ionic species supports process control in pharmaceutical, biofuel, food, and beverage industries.

Objectives and study overview

This study evaluates two high-resolution anion-exchange columns, IonPac AS11 and AS11-HC, coupled with suppressed conductivity detection to separate and quantify multiple organic acids and inorganic anions in complex yeast (Saccharomyces cerevisiae) and bacterial (Escherichia coli) fermentation broths. Key performance metrics—selectivity, detection limits, linearity, precision, stability, and recovery—are compared under gradient elution conditions.

Methodology and instrumentation

Anion-exchange chromatography with suppressed conductivity detection was performed on a Dionex DX-500 BioLC system comprising:

• GP40 gradient pump with degas option
• ED40 electrochemical detector in ASRS AutoSuppression recycle mode (300 mA)
• LC30/LC25 column oven
• AS3500 autosampler
• IonPac AS11 and AS11-HC analytical columns with corresponding AG11 and AG11-HC guard columns
• ATC-1 anion trap column to remove carbonate

Eluents were 5 mM and 100 mM NaOH prepared from 50 % (w/w) sodium hydroxide under helium blanket. Gradient programs ranged from 0.5–60 mM NaOH over 40 min with flow rates of 2.0 mL/min (AS11) and 1.5 mL/min (AS11-HC). Injection volume was 10 µL. Yeast and E. coli broths were centrifuged, heat-inactivated or diluted (10× or 100×), and directly injected without further cleanup.

Main results and discussion

Selectivity: Both columns resolved over 25 anions. The AS11-HC provided higher capacity and improved early-eluting peak resolution (lactate, acetate, formate) and tolerated larger sample loads (up to 24 µg total load) without overloading.

Detection limits and linearity: Limits of detection on AS11 ranged from 0.5 ng (Cl–) to 4 ng (BrO3–, PO43–, C6H5COO–) for a 10 µL injection. Linearity (r2≥0.999) was maintained over 0.1–12 mg/L for multiple anions and 1–1000 mg/L for selected species.

Precision and stability: Peak area RSDs were 0.4–2.1 % and retention time RSDs 0.3–0.7 % over 96 h of continuous analysis. Eluent replacement caused minor retention shifts.

Recovery: Spiking of heat-inactivated yeast broth yielded recoveries of 84–101 % for key anions.

Fermentation profiling: In S. cerevisiae cultures, lactate, acetate/glycolate, formate, valerate, methylmalonate, and citrate increased over 24 h, while phosphate decreased. In E. coli cultures, lactate decreased and acetate and succinate/malate increased.

Benefits and practical applications

• Simultaneous determination of multiple organic and inorganic anions with high sensitivity and selectivity
• Robust detection for process monitoring, yield optimization, and spoilage detection
• High-capacity column (AS11-HC) accommodates concentrated samples
• Reproducible results over extended operational periods

Future trends and opportunities

Integration with on-line bioprocess monitoring systems, automated sampling, and real-time data analysis will enhance process control. Expansion of analyte panels, miniaturized instrumentation, and coupling to mass spectrometry could extend method applicability to new fermentation platforms.

Conclusion

Ion chromatography with suppressed conductivity on IonPac AS11 and AS11-HC columns offers a sensitive, selective, and robust platform for the concurrent analysis of organic acids and inorganic anions in fermentation broths. The high-capacity AS11-HC column is particularly suited for high-load samples, supporting comprehensive bioprocess monitoring.

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