Amino Acid Analysis for Precision and Microbial Fermentation
See which amino acids your yeast, fungal or bacterial culture takes up, which it releases and how much ammonia is left, sample by sample across a run or lot by lot across your yeast extract and peptone supply.
The free amino acids in a fermentation broth show what your strain has taken up, what it has released and how much nitrogen it has left. We measure them, with ammonia, in broth samples across a run and in the complex nitrogen sources that go into the medium.
What we measure in fermentation broths
Our free amino acid profile (AA-1) covers 19 amino acids plus ammonia, reported in µM and mg/L. It uses pre-column derivatization with AQC, reversed-phase HPLC on a C18 column and UV detection at 260 nm (how AQC derivatization works). The extended panel (AA-2) adds GABA, ornithine, citrulline, taurine and hydroxyproline.
| Signal | What published work shows | Analytes to watch |
|---|---|---|
| Uptake order | In synthetic grape must, S. cerevisiae used aspartate, threonine, glutamate, leucine, histidine, methionine, isoleucine, serine, glutamine and phenylalanine early, and ammonium, valine, arginine, alanine, tryptophan and tyrosine late. Proline was not assimilated under anaerobic conditions 1. | Full AA-1 panel and ammonia |
| Preferred nitrogen | Filamentous fungi use ammonium and glutamine first; genes for secondary nitrogen sources stay under nitrogen metabolite repression 2. | Glutamine, glutamate, ammonia |
| Amino acids as carbon | In LB broth, E. coli draws its carbon mainly from amino acids recovered from oligopeptides and excretes the surplus nitrogen as ammonium 3. | Serine, aspartate, glycine, alanine, ammonia |
| Overflow | On glucose minimal media with no amino acids added, E. coli, B. licheniformis, S. cerevisiae and C. glutamicum all released amino acids at micromolar levels 4. | Valine, glycine, asparagine, lysine and others |
Uptake is sequential, so one time point rarely explains a run. A time course shows when each amino acid runs out and whether ammonia builds up late. Disappearance from the broth measures uptake, not incorporation. In wine yeast, only a limited fraction of most consumed amino acids went directly into protein; the rest was catabolized and its nitrogen redistributed 5. For grape must and wort, see wine and brewing.
Complex nitrogen sources add their own variation. Yeast extract composition can vary sharply from lot to lot with yeast strain, production process, autolysis method and purification 6. In E. coli, yeast extract lots from different suppliers, and from the same supplier, gave very different recombinant enzyme activity after induction, while respiration without induction was unaffected 6. Eight yeast extracts (six manufacturers, two lots from one) changed Azotobacter vinelandii cell dry weight up to 1.9-fold and alginate production up to twofold 7. Nine corn steep liquor batches differed in lactic acid, free amino acid profiles, total nitrogen and phosphorus, although Ogataea polymorpha proved robust to those differences 8. In yeast extracts, the reported free amino acid share ranged from 11–15% after mechanical cell disruption to 77.5% after 48 hours of autolysis at 50 °C 9. For lot checks on these inputs, see protein hydrolysates and peptones.
Questions this answers
- Which amino acids has my strain exhausted by the time growth or titer stalls?
- Is the culture secreting amino acids as feed rate or induction changes, and which ones?
- How much ammonia is left at each time point, and does it climb late in the run?
- Does this lot of yeast extract, peptone or corn steep liquor deliver the same free amino acids as the lot that worked?
- Are two suppliers' nitrogen sources interchangeable on free amino acids?
- How much free amino acid does a nitrogen supplement add to a wort or defined medium?
Caveats for this matrix
The profile keeps changing after sampling
Cells take up and release amino acids throughout a culture 1,4, and they do not stop when a sample is drawn. Handling can also move metabolites out of cells. In yeast, conventional cold-methanol quenching caused leakage that led to most intracellular levels being underestimated by at least twofold, and smaller metabolites leaked most 10. The time and handling between sampling and cell removal are part of your result. We report what is in the sample we receive, and we are glad to talk through sampling design in a free feasibility call (MD-0).
Free amino acids are not total nitrogen
Complex nitrogen sources carry much of their nitrogen as peptides. Peptides can make up more than 50% of yeast extract mass, depending on manufacture 11. In one account of autolytic yeast extract, free amino acids were 35–40% of the proteinogenic material and the rest was di- to tetrapeptides and larger oligopeptides 9. In LB, the amino acids E. coli uses come from oligopeptides 3. We measure free amino acids only; total amino acids after acid hydrolysis are not offered yet. See free vs total amino acids.
Dense broths need clarification and protein removal
High-cell-density broths carry cells, debris and secreted protein. Complete AQC derivatization needs a 4–6-fold molar excess of reagent 12, and the reaction is meant for free amino acids only. We clarify and deproteinize these samples in-house before derivatization. An internal standard goes into every sample, and every run includes a QC standard and reagent blanks (sample preparation).
Acidic or salty broths and reaction pH
AQC derivatization needs the sample buffered at roughly pH 8.2–10.1. Below 8.2 it is incomplete, and glutamic acid and alanine suffer more than serine or phenylalanine 12. Low-pH broths, or media built on corn steep liquor, which is described as rich in lactic acid 8, can pull a reaction below that range. Salts can also interfere with amino acid analysis 13. We check and adjust reaction pH, dilute where needed, and run a spike-recovery check the first time we see a matrix type (PREP-2). Results below the quantitation limit are flagged, not reported as numbers. For help interpreting a time course, see reading an amino acid profile.
Application notes
Application notes for this matrix are in preparation. The methods library covers the chemistry in the meantime.
New publications on precision & microbial fermentation analysis are reviewed monthly; relevant findings and what they change for sampling or interpretation are added to this page.
Key references
- Sequential Use of Nitrogen Compounds by Saccharomyces cerevisiae during Wine Fermentation: a Model Based on Kinetic and Regulation Characteristics of Nitrogen Permeases — Crépin L, Nidelet T, Sanchez I, Dequin S, Camarasa C. Applied and Environmental Microbiology, 2012.
- Nitrogen regulation of fungal secondary metabolism in fungi — Tudzynski B. Frontiers in Microbiology, 2014.
- Escherichia coli Physiology in Luria-Bertani Broth — Sezonov G, Joseleau-Petit D, D'Ari R. Journal of Bacteriology, 2007.
- Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms — Paczia N, Nilgen A, Lehmann T, Gätgens J, Wiechert W, Noack S. Microbial Cell Factories, 2012.
- Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae — Crépin L, Truong NM, Bloem A, Sanchez I, Dequin S, Camarasa C. Applied and Environmental Microbiology, 2017.
- Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments — Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J. Microbial Cell Factories, 2014.
- Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046 — Sparviero S, Dicke MD, Rosch TM, Castillo T, Salgado-Lugo H, Galindo E, Peña C, Büchs J. Microbial Cell Factories, 2023.
- Metabolic studies of Ogataea polymorpha using nine different corn steep liquors — Wahjudi SMW, Engel D, Büchs J. BMC Biotechnology, 2025.
- The complexity of yeast extracts and its consequences on the utility in brewing: A review — Jacob FF, Methner FJ, Michel M, Zarnkow M, Hutzler M. BrewingScience, 2019.
- Leakage-free rapid quenching technique for yeast metabolomics — Canelas AB, Ras C, ten Pierick A, van Dam JC, Heijnen JJ, van Gulik WM. Metabolomics, 2008.
- Insights Into the Complexity of Yeast Extract Peptides and Their Utilization by Streptococcus thermophilus — Proust L, Sourabié A, Pedersen M, Besançon I, Haudebourg E, Monnet V, Juillard V. Frontiers in Microbiology, 2019.
- Derivatization of Amino Acids Using Waters AccQ•Tag Chemistry — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- 〈1052〉 Biotechnology-Derived Articles—Amino Acid Analysis — United States Pharmacopeia, harmonized general chapter (official May 1, 2018).
OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Updated September 23, 2026
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