Monitoring of 20 Amino Acids and Other Key Compounds in Fermentation Processes

Importance of the topic

Amino acids are fundamental building blocks of proteins and key indicators of cellular metabolism in bioprocesses. Accurate monitoring of their concentrations in fermentation broths enables optimization of feeding strategies, supports strain engineering by revealing metabolic bottlenecks, and improves yield and quality in biotech applications.

Objectives and overview

This study describes the development and validation of a triple quadrupole LC/MS/MS method for quantifying 20 underivatized amino acids in microbial culture media. The method aims to simplify sample preparation, avoid derivatization, achieve high sensitivity and linearity, and demonstrate applicability in fed-batch and perfusion bioreactor processes.

Methodology and instrumentation

Sample preparation involves centrifugation, filtration through 0.2 µm filters, and two-step dilution in 0.1 N HCl and acetonitrile. Calibration curves (11 levels, 0.001–25 µM) were generated in 90 % ACN at pH 3. Data acquisition and optimization used Agilent MassHunter software.

Instrumentation

• LC system: Agilent 1290 Infinity Binary Pump, Multisampler, and Thermostat
• Column: Agilent InfinityLab Poroshell 120 HILIC-Z, 2.1 × 150 mm, 2.7 µm, 30 °C
• Mobile phases: A = 20 mM ammonium formate in water (pH 3), B = 20 mM ammonium formate in 9:1 acetonitrile:water (pH 3)
• Flow rate: 0.6 mL/min; injection volume: 5 µL; run time: ~18 min
• MS: Agilent 6490 Triple Quadrupole with Jet Stream ESI (Positive mode), capillary 4.0 kV, nozzle 1.0 kV, source 250 °C, desolvation 400 °C, gas flows optimized
• Detection: MRM transitions optimized for each amino acid, dwell 45 ms, EMV+: 200 V

Main results and discussion

The method achieved baseline separation of critical isobaric pairs (leucine/isoleucine, threonine/homoserine) and R² values > 0.99 for most amino acids. LODs ranged from 0.002 to 1.5 µM, LOQs from 0.006 to 3.0 µM. Intra- and inter-run precision were ≤ 20 % across concentration ranges. Retention times remained stable without additional maintenance.

Application to an engineered E. coli fed-batch culture revealed faster consumption of threonine versus methionine and significant accumulation of alanine and homoserine, indicating targets for further strain optimization. A baker’s yeast study demonstrated robustness against complex matrices and the ability to extend the panel to additional metabolites, including nucleotides and pathway intermediates.

Benefits and practical application

• Simplified sample preparation without derivatization
• Short run time (~18 min) and high throughput
• Low LOD/LOQ for accurate monitoring of trace levels
• Wide dynamic range with linear or quadratic calibration models
• Versatility for both bacterial and yeast cultures, and extension to other small molecules

Future trends and possibilities

Expanding the method to include water-soluble vitamins, nucleotides, organic acids, and signaling molecules through additional MRM segments. Integration with automated sampling and real-time process analytics will enhance feedback control. Adoption of high-resolution MS and UHPLC columns could further broaden metabolomic coverage in bioprocess development.

Conclusion

The validated LC/MS/MS approach offers a robust, sensitive, and high-throughput solution for amino acid profiling in fermentation media. Its flexibility in detecting underivatized compounds, combined with simple sample handling, supports metabolic engineering, bioprocess optimization, and comprehensive monitoring of microbial cultures.

References

1. Walther T, Calvayrac F, Malbert Y, Alkim C, Dressaire C, Cordier H, François JM. Construction of a synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine. Metabolic Engineering. 2018;45:237-245. DOI:10.1016/j.ymben.2017.12.005