Maintenance and upkeep of DO electrodes
The dissolved oxygen concentration (DO) in the fermentation broth is a crucial parameter in the process of aerobic microbial fermentation and cell culture. By observing the change of dissolved oxygen in the fermentation broth, we can know whether the growth and metabolism of microorganisms is normal, whether the process control is reasonable, whether the oxygen supply capacity of the equipment is perfect, etc.
Therefore, real-time and accurate measurement of this parameter is the basic premise of realizing automatic control of dissolved oxygen. At present, the in-vessel monitoring is mostly carried out by inserting DO electrodes in the industry.
# 1
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 KCl solution. The outer surface of the cathode is covered with a layer of oxygen-permeable film, and the film can be made of breathable materials such as polytetrafluoroethylene, polyvinyl chloride, polyethylene, and silicone rubber. A polarization voltage of 0.5~1.5V needs to be applied between the anode and cathode electrodes.
During use, 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.
The optical dissolved oxygen electrode adopts the principle of quenching of active fluorescence by specific substances in basic physics. The design of the sensor is to illuminate the fluorescent substance on the inner surface of the fluorescent cap with blue light emitted by a light emitting diode (LED), generates cold light phenomenon (red light)), by detecting the phase difference between red light and blue light, and comparing with the internal calibration value, then the oxygen flow is easily calculated, and finally, the final value is output through automatic temperature and air pressure compensation.
Note: The HOLVES bioreactor is standard equipped with METTLERTOLEDO InPro6800 series polariscopic DO electrodes. The following information is for this type of electrode only.
# 2
Preparation before use of DO electrode
- Electrode liquid
If the DO electrode is used for the first time or has not been used for a long time, it is recommended to replace the electrolyte before use. It is generally recommended that customers replace the electrolyte every three months, and the decision can be made according to the specific situation.
If the electrode signal is abnormal (such as long response time, increased current in anaerobic media, etc.) or the electrode appears "mechanical damage", it is necessary to replace the membrane or return to the original factory for maintenance.
If the electrode signal is abnormal (such as long response time, increased current in anaerobic media, etc.) or the electrode appears "mechanical damage", it is necessary to replace the membrane or return to the original factory for maintenance.
- Procedure for changing the electrolyte:
- 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 rinsing;
- Tilt the membrane, and the nozzle of the electrolyte bottle is vertically downward;
- Gently squeeze the electrolyte bottle, so that the electrolyte slowly flows into the membrane;
- The amount of electrolyte added is about 1/2;
- Confirm that there are no air bubbles inside the film, if there are air bubbles, flick the film body to remove air bubbles;
- Slowly rotate the membrane into the inner electrode, and then carefully tighten the stainless steel sleeve.
- Polarization of 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:
- When the electrode is used for the first time, it should be polarized for more than 6 hours;
- Replace the membrane or electrolyte and polarize for more than 6 hours;
- When the transmitter is powered off or the electrode is disconnected from the cable, the minimum polarization time is shown in the table below.
# 3
Calibration of DO electrode
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, first put it in an oxygen-free environment, 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 is determined by the calibration medium:
- If air is used as the calibration medium, place the electrode in the air and wipe the water off the membrane. After the reading is stable, click "full scale confirmation";
- In the process of biochemical fermentation, the 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.
# 4
Performance test of DO electrode
Each electrode has its own zero and slope, and with the prolongation of use time, the electrolyte is gradually consumed, and the slope and zero of the electrode will also change accordingly. And through the change of slope and zero, we can infer the performance of the electrode
# Slope judgment method:
After calibrating with air as the calibration medium, refer to the air current standard in the polarographic dissolved oxygen electrode current signal table to determine whether the slope of the DO electrode is normal.
If it is in the warning or alarm range, replace the electrolyte or membrane of the electrode and then re-calibrate. If it is still in the warning or alarm range after calibration, the electrode needs to be returned to the factory for repair.
If it is in the warning or alarm range, replace the electrolyte or membrane of the electrode and then re-calibrate. If it is still in the warning or alarm range after calibration, the electrode needs to be returned to the factory for repair.
# Zero judgment method:
After calibrating with nitrogen with a purity of 99.995% as the calibration medium, refer to the zero current standard in the polarographic dissolved oxygen electrode current signal table to judge whether the zero of the DO electrode is normal.
If it is in the warning or alarm range, replace the electrolyte or membrane of the electrode and then re-calibrate. If it is still in the warning or alarm range after calibration, the electrode needs to be returned to the factory for repair.
If it is in the warning or alarm range, replace the electrolyte or membrane of the electrode and then re-calibrate. If it is still in the warning or alarm range after calibration, the electrode needs to be returned to the factory for repair.
# 5
The current value of the electrode in the air is abnormal
The current value of the electrode in the air refers to the current value of the electricity exposed to the air, which is generally expressed as an absolute value. Different types of DO electrodes have different current value ranges in the air. See the electrode instruction manual for details.
# The current value in the air is low, possible causes and solutions:
- The surface of the platinum cathode is covered with oxides
In this case, when the head of the inner electrode faces the light source and observes the cathode, it can be seen that the surface of the cathode appears black. You can use sandpaper with a grade of 1000 or more to lightly sand the head of the platinum wire for several times until the surface of the platinum wire is shiny. Do not over-sand as this will damage the inner electrode head.
- The platinum cathode fails to contact the dissolved oxygen membrane
Check whether the dissolved oxygen membrane is screwed in place, if not, you must screw the membrane in place, and tighten the membrane protective cover until the black sealing ring is no longer visible.
Check the DOF membrane, if there are excessive protrusions, so that the cathode cannot touch the membrane, the DOF must be replaced.
Check the DOF membrane, if there are excessive protrusions, so that the cathode cannot touch the membrane, the DOF must be replaced.
# The current value in the air is too high, the possible causes and solutions:
| Cause | Solution |
| Electrode polarization is insufficient | Confirm whether the polarization time matches |
| Electrodes are soiled | To clean the electrode, deionized water should be used instead of cleaning solution containing ethanol |
| Electrolyte aging | Replace the electrolyte |
| Membrane aging or damaged | Replace the membrane |
| Cable damaged | Replace the cable, the transmitter should display a very low and stable current value when the electrode is not connected |
| Transmitter damaged | Replace the transmitter |
# 6
Maintenance of DO electrode
During use, inaccurate or unstable measurement is most likely to occur due to clogging of the membrane, which is often caused by the attachment of tiny ions to the membrane surface. Such clogging is generally not easy to detect with the naked eye. For this kind of pollution, the electrode can be removed and soaked in 3% to 5% dilute hydrochloric acid for several hours before use.
When the electrode is not used for a long time, the protective cap should be covered and stored in a protective box.
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