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 afterwrds 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 hihger atmospheric concentraction of 0,312 %. Besides there were no experience described in litarature 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 shwon 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 intracelluar 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 archieved 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 soulable than oxygen in water so that it must be evaluate how much carbon dioxde the cell tolerate and how much oxygen is dispensble for the implemtation 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.

Design of an appropiate process for the measurement of CO2 fixation.
Mixure of carbon dioxde and air of the exhaust gas because the Qubit analyzer is too sensitive.
Avoiding tailback by additional pump -> pulsing Figure 4

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

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.

Equation to calculate the concentration of carbon dioxide from the output of the Qubit Analyzer
x = y - 1555,34754 / 4,10739
this needs to be estimated by the portion of exhaust gas from the reactor to quantify the amount of carbon dioxide precisely

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 to find appropiate conditions for the cultivation where there is first an efficient suplly with oxygen and afterwards limitation so that the carbon dioxide fixation can be characterized and compared under both condtions ideally.

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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