The pH that you are measuring is related to dissolved concentration of CO2 in the media. During this process problably you are changing the balance of O2/CO2 and might having this result. I am not an expert on this thema, however would you reccomend to take a look at this

https://www.fondriest.com/environmental-measurements/parameters/water-quality/ph/

This is Dr. Sakthikumar Ambady answer. I hope It is useful for you.

Did you mean that your culture medium is becoming alkaline (pinkish) when stored for a longer time? That is normal. No need to worry. CO2 is your answer. The bicarbonate buffering system used in most culture media is standardized for either 5% or 10% CO2. CO2 is highly soluble in water to produce carbonic acid that ionizes to H+ and HCO3-. As you know, the amount of H+ ions determines the pH. More CO2 produce more H+ ions and hence the medium turns to acidic pH. Low CO2 will produce fewer H+ ions and hence the pH moves to the alkaline side, Atmospheric CO2 is only 0.3% and therefore fewer H+ ions and hence alkaline pH. You can keep the media bottle in the incubator with the cap slightly loose allowing CO2 to dissolve in the medium and bring up the pH to the 7.2-7.4 range. If on the other hand, media in culture plates is turning alkaline in the incubator, that tells you that you have run out of CO2 in the tank or that the CO2 sensor in your incubator is faulty.

Dear Sahil,

depending on your medium composition and/or the metabolic activity of your cells, you are probably stripping out accumulated CO2 (in form of acidifying bicarbonate and carbonate) from your culture and with that the pH of your culture increases. How significant is the pH change?

Regards,

Michael

Dear Sahil Sharma

As previous scientists said before me, CO2 stripping must induce such pH increase.

Looking at the dissolved CO2 equilibrium in water:

CO2 + H2O H2CO3 2H+ + CO32-

Sparging air engender a decrease of dissolved CO2 then reduction of free proton H+ and pH increase.

Best regards,

Paul R. Jr BROU

Dear Mr. Sahil,

It is quite normal to see pH changes with air sparging as more CO2 gets dissolved in medium which gets converted to carbonic acid (a week acid), thereby decreasing the pH of medium. However, the net change in pH does depends on the medium composition also.

Dear @Paul R. Jr Brou

How Sparging air in media lead to decrease in dissolved CO2 of the media?

Dear Sahil Sharma ,

By sparging air, you will add O2 to your medium and O2 will progressively replace CO2 in the medium.

There is a gas transfer between the liquid medium and the bubble of sparged air.

Oxygen in higher concentration in gas will spontanatly diffuse to the liquid.

Carbon dioxyde in higher concentration in liquid will spontanatly diffuse to the gas phase.

Moreover bubble going from bottom to top contribute to a faster CO2 removal from the liquid.

Hope it will help you.

Best regards,

Paul

Dear Sahil,

I think, It is not only increases, can decease also. It depends upon the bioreactors set point.

The reasonable answer is that, it is quite normal to see pH changes with air sparging as more CO2 gets dissolved in medium which gets converted to carbonic acid (a week acid), thereby decreasing the pH of medium. As part of the pH control loop, air sparging can be increase to improve CO2 removal automatically, hence increase culture pH, and vice versa.

One example of cell cultures, many therapeutic proteins are produced in mammalian cell cultures using stirred‐tank bioreactors. Those bioreactors provide excellent control of critical parameters for optimal cell growth and protein production such as temperature, dissolved oxygen (DO), and pH. Previously, it is seen that sufficient air sparge is critical in CO2 removal and gas throughput (vvm) has been used as a criterion in our routine bioreactor scale-up practices. Bioreactor pH could be effectively controlled with the utilization of sparge gas composition across a range of processes and pH conditions. Traditionally, pH control is realized through a two‐sided control loop, where CO2 sparging or base addition is used to decrease or increase pH, respectively. For example, when pH is higher than the setpoint, CO2 sparging is used to modulate the CO2‐bicarbonate equilibrium in bicarbonate‐buffered media, and reduce pH; when pH is lower than the setpoint, a pump is triggered to deliver base (e.g., sodium hydroxide [NaOH], sodium carbonate [Na2CO3], or sodium bicarbonate [NaHCO3]) to increase pH. A high amount of base addition leads to dramatic osmolality increase; and it is well‐recognized for its effect in compromising culture performance. Improving mixing and reducing base usage are often pursued for process improvement.

Ashish