The pH value of the fermentation medium has a very obvious influence on the growth of microorganisms, and is also an important factor affecting the activities of various enzymes in the fermentation process. Therefore, the monitoring and adjustment of pH is very important in the fermentation process.
During the fermentation process, the pH is usually monitored in real time by directly inserting the composite pH electrode into the fermentation liquid in the tank. The high pressure and high temperature sterilization operation and the physical and chemical properties of the fermentation broth will affect the pH electrode measurement, so the correct use method and daily maintenance are particularly critical.
1.Preparation before installation
1)When unpacking, carefully inspect the pH-sensitive membrane glass, diaphragm (bisquette ceramic core), and glass body of the electrode for mechanical damage.
2)Remove the liquid container and wash the top of the electrode with pure water, then gently dry it with a wet tissue or absorbent paper. Be careful not to rub the pH-sensitive membrane to increase the response time.
3)Gently move the pH electrode to a vertical position to prevent air bubbles in the pH-sensitive membrane glass bulb. If it is not filled with liquid or there are air bubbles, gently shake the electrode to fill the bulb with liquid until there are no air bubbles.
4)The electrode can be soaked in acidic buffer (pH 4.01) for several minutes before use, rinse the glass bulb with pure water, and then use absorbent paper to gently absorb the water in the glass bulb, and then put it in neutral buffer. (pH6.86 or 7.00, etc.) for a few minutes to activate the electrode, and then start the calibration.
2.pH electrode two-point calibration operation
Soak the pH electrode in the standard buffer for 10min, and after the measured value is stable for about 1min, then perform the first point calibration and the second point calibration of the pH electrode in sequence.
Take the Biorectek™ bioreactor fermenter as an example:
1）Before calibration, make parameter selection based on buffer type:
[GB] refers to the use of buffers that meet the GB/T 27501-2011 standard. The pH values of several buffers generally used are 4.00, 6.86 and 9.18, and the corresponding “stability” is “the uncertainty of the buffer. degree” is usually selected as ±0.02pH.
Bioreactek usually uses the METTLER TOLEDO InPro3030 series pH electrode. The parameter [MT_9] corresponds to its brand of buffer solution. The pH values of the buffer solution generally used are 4.01, 7.00 and 9.21, and its “stability” depends on the buffer solution used. model to select.
2）Connect the electrode, rinse the electrode with pure water, and then gently dry the water on the probe with absorbent paper.
3）Partially immerse the glass bulb in the first buffer solution (eg pH=4.01) (the diaphragm should be completely immersed in the buffer solution), and click the first point to confirm after the standard value is stable (30 seconds to 60 seconds). Point calibration ends.
4）Remove the electrode from the first buffer and rinse the electrode with pure water, then gently blot the water on the probe with absorbent paper.
5）Partially immerse the glass bulb in the second buffer solution (eg pH=9.18) (the diaphragm should be completely immersed in the buffer solution), click the second point to confirm after the standard value is stable (30 seconds to 60 seconds), and the second When the calibration is over, wait for it to be used (it is recommended that the time should not be too long).
3.Precautions for Electrode Calibration1)
1）Be careful to use fresh buffers when calibrating 😉
2）The electrode is placed in the buffer for 1 min before subsequent operations;
3）After rinsing the electrodes, only use a soft absorbent paper to dry up the water, never rub the pH-sensitive membrane;
4）The calibration cycle of the electrode is determined according to different use environments and accuracy requirements. Please determine the appropriate calibration cycle under the premise of ensuring accuracy;
5）Since the pH electrode probe is extremely fragile, do not bump it during use.
4.pH electrode performance test
The pH electrode measurement method is based on the principle of Nernst equation. The electromotive force of the electrode has a linear relationship with the pH value. Generally, two buffer solutions with different pH values are used for calibration to determine the slope of the curve. The so-called pH electrode response slope refers to the pH electrode used to convert the millivolt (mV) signal of the electrode into a pH value. It is the voltage difference measured by different buffers and divided by the buffer difference. of. This slope is an important indicator to determine whether the electrode life is exhausted.
It should be noted that since the slope is proportional to the temperature, when the solution temperature changes, according to the Nernst equation, the ΔE of the solution will change linearly with the temperature T, and the electrode is converted into pH according to the detected electromotive force of the solution. value, so temperature compensation must be performed to offset the effect of temperature on the measurement results.
The slope is proportional to the temperature
The so-called temperature compensation is to convert the slope obtained by the electrode at the calibrated temperature (usually 25°C) to the slope at the current temperature according to the Nernst formula, so as to obtain the correct pH value at the current temperature. It is mainly used to correct the deviation caused by the difference between the temperature of the standard sample such as standard buffer and the actual sample solution temperature during calibration.
Bioreactek series products can measure the current liquid temperature through the temperature electrode of the device, and then display the pH value after temperature compensation after calculation by its own software. Therefore, whether it is calibration or performance testing, it is necessary to ensure that the temperature electrode of the device is in working condition.
Slope test specific operation method:
1)Wash the electrode after two-point calibration with pure water, and use a soft absorbent paper to absorb the water.
2)Adjust the parameters and stability according to the method used in the calibration above. The MT standard is used as an example below.
3)First, use pH=7.00 buffer to measure the zero point, and read the mV value on the display. Bioreactekstandard pH electrode zero point is in the range of 6.5~7.5, indicating that the electrode is normal.
4)After cleaning the electrode, insert it into the standard buffer solution with pH=4.01 (referred to as pH1), and read the mV value (referred to as mV1) on the display screen.
5)After cleaning the electrode, insert it into the standard buffer solution with pH=9.21 (referred to as pH2), and read the mV value (referred to as mV2) on the display screen.
6)Calculate the slope of the electrode, namely (mV1-mV2)/(pH1-pH2).
7)According to the Nernst equation in an ideal state (25°C), the ideal slope is 59mV/pH, that is, every time the pH value of the solution changes, the electrode will produce
8)59mV/pH or so. When the value of the slope is less than 53mV/pH or someone is at 63mV/pH, a new pH electrode needs to be replaced, so when the calibration slope is in the range of 53~63mV/pH, the result is credible.
The Bioreactek series fermenter can directly read the voltage signal of the liquid measured by the electrode, and if there is a problem with the electrode or the installation or use is wrong, a red prompt message “electrode unavailable” will pop up at the bottom of the pH calibration interface, which is convenient for customers to understand the use status of the electrode.
5.Cleaning of electrodes1)
1)For general contamination, wash the electrode with water, 0.1mol/L NaOH or 0.1mol/L HCl for several minutes.
2)Grease or organic contamination Clean the electrode with acetone or ethanol for a few seconds.
3)Sulfide pollution (diaphragm blackening) Treat with thiourea/HCI, soak the glass bulb part in the solution (the diaphragm should be submerged in the solution) until the diaphragm is colorless (at least 1 hour), then soak in 3mol/L KCI for at least 12 hours, fully rinsed and recalibrated for use.
4)Protein contamination (yellowing of the diaphragm) was treated with gastrin/HCl, the glass bulb section was placed in the solution, ensuring that the diaphragm was submerged in the solution (at least 1 hour), then rinsed with distilled water, recalibrated.
Rinse the pH electrode Soak the pH electrode
6.Storage of electrodes
1)After each production cycle, carefully rinse the electrode tip and diaphragm with deionized water, never allow the measurement solution on these parts to dry out.
2)The electrode cannot be stored in distilled water. When not in use for a long time, it should be fully immersed in 3mol/L KCI or 9816/ViscolytTM electrolyte along with the electrode tip and diaphragm.
3)The electrode cannot be stored dry for a long time, and the electrode cannot be stored with a dry medium attached to the surface. If the electrode is stored dry by mistake for several days, it should be soaked in normal storage electrolyte for several hours before use.
4)Connectors should be checked from time to time for signs of moisture. If necessary, rinse thoroughly with deionized water or alcohol, then dry carefully.