Cultivation

The aim of the cultivation was to characterize the carbon dioxide fixation in E. coli by an appropriate process. For this approach we wanted to establish a fermentation process, where E. coli is growing first under aeration to obtain aerobic growth conditions to determine the carbon dioxide fixation of the RuBisCo and the Carboxysome afterwards under this condition, as well as conditions were oxygen is limited, but still aeration takes place. Besides we wanted to ensure that an effective carbon dioxide fixation would be measured. Therefore we gased in additional carbon dioxide, resulting in an 10 fold higher atmospheric concentration of 0,312 %. Besides there were no experience described in literature how much carbon dioxide can be used by a hetereotrophic bacteria like E. coli. To determine even the slightest change in the carbon dioxide balance a the very sensitive Qubit Analyzer was used for the carbon dioxide fixation...
Besides the complex equilibrium of carbon dioxide (CO₂) and bicarbonate (HCO₃^-) as shown in Figure 1 have to be considered. One aspect is that the higher concentration of carbon dioxide is inhibiting hte bacterial growth by stressing the cell to maintain the intracellular pH value and decreasing the pH optimum of its enzymes when the cell is not able to regulate the pH anymore. A higher concentration of carbon dioxide (CO₂) is archived by excess pressure, a higher stirrer speed or lower pH value. This parameter are also optimal for an efficient oxygen supply of the cell, but the problem is that carbon dioxide is better soluble than oxygen in water so that it must be evaluate how much carbon dioxide the cell tolerate and how much oxygen is dispensable for the implementation of the process.

Figure 1: Carbonate equilibrium. For an optimal process a consideration of how much carbon dioxide can be tolerated by the cell is needed.

For the design of an appropriate process for the measurement of CO2 fixation the system shown in Figure 2 was established. In this system the reactor is gased in with a mixture of carbon dioxide, in a total amount of 0,312% and air. The flow rate was set up and verified to 334,26 ml/min.
Because there are no reported experience how much carbon dioxide can be used by an organism which is normally not able to use carbon dioxide, but produce it, the very sensitive Qubit Analyzer was used for the quantification of carbon dioxide. But because of its sensitivity the exhaust gas of the reactor needs to be diluted, to avoid an overload of the Qubit Analyzer. This is realized by another gas mixture with air, so that only 4% of the exhaust gas of the reactor are analyzed in the Qubit Analyzer. But as shown in the final cultivation process the measurement is still very sensitive and the calibration was also possible without some fluctuations due to the reduced amount of analyzer.
To obtain an constant flow to the Qubit Analyzer and excess pressure of the reactor is necessary, and also a pump must be established between the displacer of the 4% of the exhaust gas and the reactor avoiding tailback of the pressure into the reactor and unstable growth conditions. The same problem occurs afterward when the flow rate needs to be limited for the Qubitr Analyzer to 150 ml/min, while for the gas mixture higher flow rates need to bee performed. Therefore gas outlet was established to avoid a second tailback. Apart the cool traps are used for dehydration of the exhaust gas, because the measurement of carbon dioxide within the Qubit Analyzer is performed within infra red detection and therefore dry gas are needed. Additional to the cooling traps a dimerite column was established just in front of the Qubit Analyzer to absorb the remaining water of the gas.

Figure 2: Schematic construction of the reactor for the measurement of carbon dioxide fixation in the hetereotrophic bacteria E. coli.

The calibration was performed with different concentrations of carbon dioxide (compare Figure 4 and 5), while the zero set point of 0% CO₂ was reached with a wash column of NaOH and Calciumhydroxide (Ca(OH)₂), which absorbs the carbon dioxide to Natriumcarbonat (Na₂CO₃).

Figure 3: Calibration 10% of the exhaust gas (no pulsing). Higher concentration of the fermentation could not be recorded with this high portion of exhaust gas from the reactor.

Figure 4: Calibration 4%, which where used for establishment of the final process and the calibration in Figure 5.

Figure5x: Calibration by linear fit of the output signal of the qubit analyzer to determine the carbon dioxide fixation.

The linear fit of the calibration yield in the equation for the calculation of the carbon dioxide concentration from the output of the Qubit Analyzer and could be determined as:
x = y - 1555,34754 / 4,10739
The equation can be used for the determination of the portion of exhaust gas from the reactor to quantify the amount of carbon dioxide precisely, by multiplication of 4%.
As the estimation shows the same results then the measurement (Figure 6) the system seems to work accurate!

Figure 6: Comparision of the calibration by the measured carbon dioxide using the Qubit Analyzer and calculation from the measured flow rates of the system.

Long time process optimization were performed to find appropriate conditions for the cultivation, where there is first an efficient supply with oxygen and limitation of oxygen afterward so that the carbon dioxide fixation can be characterized and compared under both conditions ideally. For this approach we have identified an inocolum of OD₆₀₀ = 0.03 as ideal, ensuring a longer time under aerobic growth conditions. A pH-value of 7,1 allowing relative weak fluctuations in the carbon dioxide signal, compared to lower pH values as one aspect. And reducing the additional salt load in comparison to higher pH values as the other aspect. Beside the excess pressure was kept by 0,1 mbar to obtain enough pressure for the displacer of the exhaust gas and avoiding to much pressure for a higher solubility of carbon dioxide resulting in a longer lag-phase because of the intracellular stress. The stirrer speed of 600 rpm was turned out to be usable for aerobic conditions, resulting also in oxygen limitations after some time. But a faster oxygen limitation is probably desirable and can be archived simple be the reducing of the stirrer speed. Apart the gas entry of carbon dioxide and oxygen was kept constant in all experiments 334,26 ml/min to obtain stable conditions of the gas mixture as the gas entry can be affected by other parameters like the stirrer speed or the excess pressure. Furthermore the temperature of 37 °C was never changed to obtain optimal growth conditions of E. coli.
After the development of the cultivation process for the measurement of carbon dioxide fixation in a hetereotrophic bacteria, the process shown in Figure 7 shows the desired requirements for the quantification, as there is first a phase of full oxygen supply, which decreases in the exponential phase with the bacterial growth to end up in the desired oxygen limitation! With this system a quantification of the carbon dioxide fixation of a organism that is normal not able to use carbon dioxide as a substrate in quantitative amounts should be able up to now!

Figure 7: Successfull cultivation process for the measurement of carbon dioxide.

References

Sage, Rowan F. „Variation in the K(cat) of Rubisco in C(3) and C(4) Plants and Some Implications for Photosynthetic Performance at High and Low Temperature“. Journal of Experimental Botany 53, no. 369 (2002): 609–20.
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