Cations and anions
Fermentation broths typically have several cations and anions, including amino, carboxylic and other organic acids, as well
as amines and other organic bases. However, concentrations of inorganic cations and anions are significantly higher than their
organic counterparts. Thus, inorganic ions show significant peaks by IC when fermentation broths are diluted 1000-fold or
more, while their organic counterparts are reduced to undetectable levels. This provides necessary selectivity for analysis.
The first report of the determination of cations and anions using IC to follow a fermentation process of methanotrophic bacteria
was published in 1992 (7). Using OmniPac PAX-500 anion-exchange column and SCD, the authors followed several anions over the
course of fermentation and found acetate to accumulate when natural gas was used as the substrate, which inhibits the growth
of the methanotrophic bacteria. Mono- and divalent cations were monitored by SCD using IonPac CS10 cation-exchange column
eluted isocratically with 40 mM HCl–4 mM DL-2,3-diaminopropionic acid (DAP) as the eluent. The results indicated that the
concentration of Mg2 + must be above 60 ppb to maintain optimum growth of the bacteria.
Robinette et al. (8, 9) reported quantitation of eight mono- and divalent cations in fermentation broths for recombinant and
pathogenic microorganisms, as well as in mammalian and insect cell cultures in complex and defined media. The authors employed
IonPac CS10 and CS12 cation-exchange columns eluted isocratically with 20 mM HCl – 4 mM DAP and 20 mM methanesulfonic acid
(MSA), respectively. The cations are detected by SCD. The sample preparation involves simple 1000–2500 fold dilution of filtered
broth with water. The reports indicate good precision and accuracy, and a wide linear range for each cation, with detection
limits ≤1 µg/mL.
Figure 1: A time course of analysis of cations in an insect cell culture using a complex medium with glucose and sucrose as
the primary carbon sources. Peaks: 1=sodium, 2=ammonium, 3=potassium, 4=magnesium, and 5=calcium. Column: IonPac CS12; elution:
20 mM MSA, isocratic, 1.0 mL/min. (Reprinted from Ref. 9 with permission from Elsevier)
Figure 1 shows a time course of an insect cell culture using a complex medium. The chromatograms show that the levels of sodium,
potassium, magnesium, and calcium remained virtually unchanged throughout the fermentation, while ammonium concentration increased
approximately 2-fold. Similar investigations were reported for S. cerevisiae (eukaryotic) grown in a chemically defined medium, and
(prokaryotic) and a mammalian cell culture in complex media containing glucose as the primary carbon source.
Figure 2: Resolution of carbohydrates, alditols, alcohols, and glycols commonly found in fermentation broths by (A) CarboPac
MA1 column and (B) CarboPac PA1 column. (Reprinted from Ref. 12 with permission from Dionex)
Aliphatic carboxylates (e.g., acetate, lactate, pyruvate) are metabolites that often reduce fermentation yields. In an Application
Note (10), Dionex reported the analysis of aliphatic carboxylates and inorganic anions in the fermentation broths of S. cerevisiae in YPD medium and
in LB medium using IonPac AS11-HC column eluted with 1-60 mM NaOH and SCD. The results show well resolved peaks with good
precision and accuracy, wide linear range and low detection limit for each anion.
Figure 3: A time course of analysis of carbohydrates in H. influenzae cultivation using complex medium with glucose as the
major carbon source. Peaks: 1=glucose, 2=fructose, 3=ribose; 4=maltose. H=hours. Elution: 150 mM NaOH, isocratic, 1.0 mL/min.
(Reprinted from Ref. 11 with permission from Elsevier)
Carbohydrates and alcohols
Carbohydrates are carbon sources essential for cell growth and product synthesis, while alditols and alcohols are metabolites.
High Performance Anion-Exchange Chromatography (HPAEC) coupled with PAD (HPAEC–PAD) or Integrated PAD (IPAD), which is an
improved PAD application, is the most widely used chromatographic technique for the analysis of carbohydrates and alditols
(3–6). The CarboPac MA1 column has the ability to analyze mono- and disaccharides, alditols, and alcohols present in fermentation
broths (see Figure 2A) in a single run. However, the chromatography takes as long as 60 min for each run and uses a high concentration
of NaOH as the mobile phase. The CarboPac PA series of columns (e.g., PA1, PA10, PA20) take significantly less time and use
low NaOH concentration. But alcohols and alditols are eluted early and are poorly resolved (Figure 2B). Therefore, the PA
series columns are better suited for mono- and disaccharide analysis. Thus, the choice of column depends on the analytes to
be monitored in the media. Herber and his colleagues (8, 11) used a CarboPac PA1 column to monitor microbial fermentations
using chemically defined and complex media. The samples were diluted 50 fold with water and eluted from the column with NaOH
(isocratic and gradient) to monitor ethanol, glycerol, galactose, glucose, mannose, fructose, raffinose, ribose, and lactose.
Carboxylic acids and inorganic ions are transparent to PAD. Most of the other media components were below the detection limit
at this dilution. Indeed, only proline, arginine and lysine exhibited any noticeable detector response (see later for the
detection of amino acids by PAD). Figure 3 shows a time-course study of carbohydrates in a H. influenzae fermentation in a complex medium with glucose as the major carbon source. Figure 4 shows the chromatograms of the analysis
of S. cerevisiae fermentation broth using CarboPac MA1 (12). Glucose is the most predominant component of the media at 0 h. At the end of fermentation,
no glucose was detected and the predominant components are the metabolites.
Figure 4: Chromatograms of the analysis of S. cerevisiae fermentation broth using CarboPac MA1; (A) 0 h and (B) 24 h. (Reprinted
from Ref. 12 with permission from Dionex)