The dissolved oxygen concentration in the fermentation broth (Dissolved Oxygen for short DO) is a crucial parameter in the process of aerobic microbial fermentation and cell culture. , By observing the changes of dissolved oxygen in the fermentation broth, we can know whether the growth and metabolism of microorganisms are normal, whether the process control is reasonable, and whether the oxygen supply capacity of the equipment is perfect.
Therefore, the real-time accurate measurement of this parameter is the basic premise to realize the automatic control of dissolved oxygen. At present, most of the in-tank monitoring is carried out through the plug-in DO electrode in the industry.
- Basic types of DO electrodes
Two kinds of dissolved oxygen electrodes are commonly used in the fermentation industry – polarographic dissolved oxygen electrodes and optical dissolved oxygen electrodes.
The polarographic dissolved oxygen electrode is made of platinum (or gold ring) as the cathode and silver-silver chloride (or mercury-mercurous chloride) as the anode. The electrolyte is KCI solution. The outer surface of the cathode is covered with a layer of oxygen permeable The film can be made of breathable materials such as polytetrafluoroethylene, polyvinyl chloride, polyethylene, silicone rubber, etc. A polarization voltage of 0.5~1.5V needs to be applied between the anode and cathode electrodes.
During use, the dissolved oxygen will be ionized when it reaches the cathode surface through the membrane, and the electrons released during this process will form a current in the electrolyte, because the dissolved oxygen content through the membrane is proportional to the dissolved oxygen content in the water. , so under different dissolved oxygen content, the current intensity formed in the electrolyte is also different, and the magnitude of the current intensity can be monitored by the electrode. The current intensity monitored by the electrode can be converted into a specific oxygen concentration according to Faraday’s law, and the value is obtained and then the final value is output through temperature and air pressure compensation. Since the electrolyte participates in the reaction throughout the process, the electrolyte needs to be replaced regularly.
optical dissolved oxygen electrode is based on the quenching principle of active fluorescence by specific substances in physics. The design of the sensor is to illuminate the fluorescent material on the inner surface of the fluorescent cap through the blue light emitted by a light emitting diode (LED). The phase difference is compared with the internal calibration value, the oxygen concentration can be calculated, and finally the final value is automatically compensated by temperature and air pressure.
Note: Bioreactek™ bioreactors come standard with METTLER TOLEDO InPro6800 series polarographic DO electrodes, and the following content is only for this electrode.
- Preparation before use of DO electrode
#Electrode solution
For DO electrodes that are used for the first time or have not been used for a long time, it is recommended to replace the electrolyte before use. Generally, it is recommended that customers replace the electrolyte every three months, which can be determined according to the specific situation.
If the electrode signal is abnormal (such as a long response time, an increase in current in an oxygen-free medium, etc.) or the electrode is “mechanically damaged”, the membrane needs to be replaced or returned to the original factory for maintenance.
#Operation steps for replacing the electrolyte:
1）Pour off the residual electrolyte in the membrane, rinse the inside of the oxygen-dissolved membrane with deionized water, and then use absorbent paper to absorb the water marks after the rinsing is completed;
2）Tilt the membrane, and the nozzle of the electrolyte bottle is vertically downward;
3）Gently squeeze the electrolyte bottle so that the electrolyte slowly flows into the membrane;
4）The amount of electrolyte added is about one-half;
5）Confirm that there are no air bubbles inside the film, if there are air bubbles, flick the film body to remove air bubbles;
6）Rotate the membrane slowly over the inner electrode and carefully tighten the stainless steel sleeve.
Polarization of the #DO electrode
The dissolved oxygen electrode must be electrified for more than 6 hours before use. Connect the electrode and the transmitter through a cable, and the electrode starts to polarize when the transmitter is powered on.
Electrodes need to be polarized in the following cases:
1）The electrode is used for the first time, polarized for more than 6 hours;
2）Replace the membrane or electrolyte and polarize for more than 6 hours;
3）When the transmitter is powered off or the electrode is disconnected from the cable, the minimum polarization time is shown in the table below.
| Power outage time T1(min) | Minimum required polarization time (min) |
| T>30 | 360 |
| 30>T>15 | 6*T1 |
| 15>TI>5 | 4*T1 |
| T1<5 | 2*T1 |
(DO electrode polarization schedule)
- Calibration of DO Electrodes
DO electrodes must be fully polarized before calibration. The two-point calibration used by the DO electrode needs to be operated according to the specific situation, connect the temperature electrode, and set the standard atmospheric pressure to 1013mbar.
#If there are relevant conditions, please do the calibration as follows:
After the electrode is powered on, put it in an oxygen-free environment first, click “Zero Confirmation” after the reading is stable , and then put the electrode in a pure oxygen environment, click “Full scale confirmation” after the reading is stable , and a pop-up window “DO Electrode OK” means the calibration is complete.
#If there are no relevant conditions, please do the calibration as follows:
Do not connect the electrode, click “Zero Confirmation” , the full scale calibration method depends on the calibration medium:
1）If air is used as the calibration medium, place the electrode in the air and wipe off any water marks on the membrane. After the reading is stable, click “full scale confirmation”;
2）In the process of biochemical fermentation, a saturated medium is generally used as the calibration medium. After the actual elimination and before the inoculation, turn on the stirring to the maximum at a suitable temperature, and at the same time pass in the saturated air with the maximum ventilation for a certain period of time, and click “full confirmation ” after the reading is stable . Calibration after a uniform aeration time is recommended to unify saturation across batches and fermenters.
