Figure 1: The IS-SP integration system allows operators to mount sensor spots to the inside wall of transparent containers. Here it is used to integrate a SP-CD1 carbon dioxide sensor in a shake flask.

Carbon dioxide (CO₂) is an important parameter in microbial cultures because it can inhibit or stimulate growth under certain conditions. In our experiment, we monitored Escherichia coli diauxie growth phases online and focused on dissolved CO₂ (dCO₂) and oxygen readings. We assessed diauxic growth in medium containing glycerin and glucose online with the SFR vario system (from PreSens), which optically measures oxygen, pH, and biomass in an Erlenmeyer flask. The shake flask contained an oxygen sensor spot and an optical CO₂ sensor to evaluate sensor performance for online dCO₂ monitoring in microbial culture. We determined pH, substrate concentrations, and optical density with offline measurements. Online readings showed the characteristic course of diauxic E. coli growth.

Oxygen and CO₂ measurements displayed reverse courses, as expected, indicating high oxygen consumption and simultaneous CO₂ production during metabolic activity. But in phases of reduced or stopped growth, the dissolved oxygen (DO) content rose, and the dCO₂ level dropped. Online biomass measurements were in good accordance with offline measured OD values. Compared with gas analysis (BlueSens system), DO and dCO₂ levels recorded with the SFR vario system showed higher concentration variations.

We evaluated an SP-CD1 optical CO₂ sensor spot (from PreSens) that was read out with the SFR vario device for its suitability in online monitoring of microbial cultures. Like the oxygen sensor, the sensor spot was integrated into the shake flask and read out with the reader optics typically used for online pH monitoring. The measurement system allows enables online monitoring of several parameters simultaneously. In addition to measuring biomass, DO, and dCO₂ online, we determined pH, substrate concentrations, and OD₄₂₀ offline. We compared oxygen and CO₂ levels to measurements in identical conditions taken with the BCpreFerm (BlueSens) gas analyzer for shake flasks. Measuring several parameters (all showing the same characteristics) enhanced measurement security.

Materials and Methods
We cultured E. coli MG1655 in a chemically defined medium according to Evans (1), with 0.15% glucose and 1 % glycerin as sole carbon sources. We used phosphate buffer to obtain pH 7 medium.

Figure 2: SFR vario system with sensor flask; how optical biomass measurement through scattered-light detection works: A scattered light signal is made of light reflected by the flask bottom by cells inside a medium and by reflectance at the medium’s surface.

We used 500-mL baffled plastic shake flasks with septum and a 200-mL working volume for cultivation. Flasks were equipped with an oxygen sensor spot (SP-PSt3, PreSens). The CO₂ sensor spot (SP-CD1, PreSens) was mounted at the flask bottom with an integration system (IS-SP, Figure 1) supplied by the manufacturer. The SFR vario system reads both sensors through the flask bottom, and the reader detects biomass using scattered light (Figure 2). We placed a CO₂ sensor where a pH sensor typically is integrated in sensor flasks so that the optics for pH monitoring could read it. We incubated the cultivation at 37 °C and 200 rpm. Online measurements with the SFR vario system were supported by offline monitoring of pH, glucose, and glycerin concentration using respective kits from Megazyme and determination of OD₄₂₀.

We measured oxygen and CO₂ in the gas phase of E. coli culture under the same conditions with the BCpreFerm system, a monitoring device for measuring culture headspace. That system has a larger CO₂ measurement range than the SFR vario system and is insensitive to fluorescent components in the medium that might affect optical sensors used with the SFR vario system.

Results

Figure 3: Diauxic E. coli growth on glucose (Glc) and glycerin (Gly) in 500-mL Erlenmeyer flask; biomass was determined online with the SFR vario (blue line) system and offline by OD₄₂₀ measurements. Oxygen and carbon dioxide were measured online with optical sensor spots and readout with a SFR vario system; pH, Glc, and Gly were determined offline. Two growth phases (first on Glc and then on Gly) can be determined. While oxygen is consumed during high metabolic activity, CO₂ is produced. The short lag phase at ∼4.5 h indicates that the preferred substrate has been consumed, so cells switch to metabolizing the second substrate.

Figure 3 shows both online data recorded with the SFR vario system and offline measurements. The graph shows the expected, characteristic course of diauxic E. coli growth. In the first growth phase (∼1–4 hours), glucose is a carbon source, and glycerin is not metabolized. During this phase, biomass rises, oxygen is consumed, and CO₂ is produced. Oxygen consumption and simultaneous CO₂ production indicate the aerobic metabolization of glucose. After ∼4.5 h, DO level rises again temporarily, and dCO₂ decreases. In this phase, the preferred substrate (glucose) is completely consumed, and the cells enter a short lag phase. After a few minutes, the microorganisms switch to metabolize glycerin and continue growing. DO decreases with increasing cell numbers and drops to zero level during this second growth phase. That also is displayed in a slower rise of online biomass and offline OD measurement values. Because of limited oxygen, E. coli cells can no longer grow at their maximum growth rate on glycerin. Their slowed-down growth is reflected in the slight bend of the dCO₂ curve around 6 h process time.

Figure 4: Diauxic E. coli growth on glucose and glycerin in 500-mL Erlenmeyer flask; oxygen and CO₂ in gas phase were monitored with the BlueSens system. The metabolic switch from glucose to glycerin after ∼4.5 h is shown as a small peak in the O₂ values as cells briefly stop metabolic activity.

One example shows how different parameters measured simultaneously lead to higher reliability of our process understanding. After ∼10 h, the culture enters stationary phase, although glycerin is not completely consumed at this stage. Without substrate consumption, the DO level slowly rises back to 100%, whereas dCO₂ slowly decreases. In this experiment, determining the growth rate based on online biomass signal and OD₄₂₀ values can be difficult. However, also measuring dCO₂ allowed us to see slowed down growth after an initial rapid growth phase on glycerin. We expected a drop in dCO₂ concentration toward zero at the end of cultivation. But we detected only a slow decrease to ∼1.8% CO₂, a very slow progression of O₂ and CO₂ toward initial values in both liquid and gas phases (Figures 3 and 4). That result might be caused by a slow gas exchange through the shake flask lid and oxygen limitation (from insufficient k_La of the used shake-flask setup). Another possible explanation for further CO₂ production is residual activity caused by further consumption either of products that accumulated during the growth phase or of parts of lysed cells.

BlueSens measurements during the gas phase (Figure 4) showed no large concentration changes for O₂ and CO₂, which was not the case for the dissolved parameter measurements taken with the SFR vario system. Here, O₂ levels varied only in a 3% range and CO₂ levels for only 1.5%. Oxygen consumption and correlating CO₂ production also showed in reverse courses of both graphs (Figures 3 and 4).

The metabolic switch after 4.5 h is visible as a small peak in the O₂ readings, which is displayed more clearly in the dissolved-parameter measurements recorded with the SFR vario system. In the next metabolic phase, gaseous CO₂ levels show a different course than the dissolved CO₂ readings. In the headspace, the CO₂ values drop 1% over the next 4–5 h, and the dCO₂ readings indicated further CO₂ increase in the medium. The oxygen sensor used with the SFR vario system provided exact DO measurements, and the dCO₂ readings of the integrated CO₂ sensor spot showed an exact reverse course. So the dissolved parameter readings seemed conclusive.

Expedited Culture Monitoring
Our evaluation of CO₂ sensors for microbial culture monitoring showed comprehensive results that were in accordance with expected dCO₂ development during diauxic growth and indicated cell activity levels very well. We measured O₂ levels and verified CO₂ readings. Both parameters showed according tendencies. As we expected, comparison with the established BlueSens BCpreFerm system showed that O₂ and CO₂ concentration changes in gas phase were not as distinct as those recorded in liquid we monitored Escherichia coli diauxie growth phases online and focused on dissolved CO₂ (dCO₂) and oxygen readings.phase. A similar metabolic phase pattern and reverse O₂ and CO₂ courses as with the SFR vario system were recorded. Simultaneous online monitoring of several parameters with the SFR vario system enhanced measurement security because all recordings showed the same characteristics, which allowed more precise assessment of current culture status. For this study, it was advantageous that the SFR vario system could be adjusted to read out the CO₂ sensor instead of a pH sensor. That did not require a hardware change, but only a settings update. So the online monitoring device saved time for culture monitoring in shake flasks.

Reference
1 Evans CGT, Herbert D, Tempest DW. Chapter 13: The Continuous Cultivation of Microorganisms 2: Construction of a Chemostat. Meth. Microbiol. 2, 1970: 277–327; doi:10.1016/S0580-9517(08)70227-7.

Helga Tietgens is a technical assistant at Max Planck Institut für Dynamik komplexer technischer Systeme, Magdeburg, Germany. Corresponding author Gernot T. John is director of marketing and innovation at PreSens Precision Sensing GmbH, Regensburg, Germany. Katja Bettenbrock is head of experimental systems, biology team, at Max Planck Institut für Dynamik komplexer technischer Systeme.