During cascading events of a fermentation, agitation and aeration are at a max. Thus the saturation of air with those conditions give a higher DO value compared to the saturation using basic conditions before inoculation.
Hi Vishnu,
Normally, 100% calibration should be done after sterilization of your medium and after it cooled down to your desired fermentation temperature. At that level, you can inject your air and/or oxygen and stirr properly until the current value (mA) is stable. Once it is done, you calibrate your 100%.
Peers answers/comments are welcome on this topic, i'd like to go deeper in its understanding.
Amine
Yes sir, I completely agree. But I would like to add that the highest oxygen saturation of the media will be at the highest agitation and maximum oxygen conc. in the gas mixture. Why cant it be set using these conditions and then returned to the start?
Hi Vishnu,
According to my experience, reaching 100% DO does not require necessarly reaching highest levels of stirring and aeration (i.e. maximum capacities of your fermenter). This will depend on the fermentation medium. If it is a high suspended solids medium (case of the many non synthetic media) then O2 transfer would be hampered. In this case, one may need higher stirring/aerating rates to reach saturation O2 concentration (at extreme cases, could this [O2]sat never be reachable? maybe). Otherwise one just need to set RPM and VVM at «reasonable» values and wait until DO is stable. You can make the experience to increase incremently RPM / VVM and see for which values you will reach max DO concentration. If you are using a synthetic/semi synthetic culture medium with low SS, you'll see that you reach 100% DO quickly without going too high with RPM and VVM. Then you can inoculate by keeping these RPM/VVM values.
Hope it will help you
All the best
Amine
Hi Vishnu,
I do it in this sequence, cleaning, polatization for minimum 6 hours, media sterilization, tuning initial parameters (aeration, agitation, pH, etc), stabilization of the set parameters, and finally 100% calibration of pO2 probe. I have experienced good response. Thanks
Setting of 100% oxygen saturation is performed by the user to see the rate at which oxygen is consumed and also to maintain a condition. Normally the procedure to set-up 100% initial saturation is to calibrate the probe based on the required initial conditions at which the reactor will be run before inoculation. The 100% is an empirical number here, based on which the user can regulate the dissolved oxygen as the culture grows. But in normal practice the 100% saturation is always set at the initial conditions used by the user and not at maximum condition of the rector.
The calibration technique is a standard and typically is done after sterilization before inoculation at 1 vvm of air supply and at the maximum rpm used in the fermentation (or 400-500rpm is enough),after DO is stable, around 15 min, the stable value is 100%, what important for this procedure is back pressure that typically is set-up at the value of the fermentation process, typically 0.5 bar.
The DO,% is relative value and 100% is equal to the soluble concentration, mg/L, that can be found according to Henry Law, and it depends on temperature, oxygen concentration in the supply gas (in air it is constant, around 21%) and back pressure.
Therefore if your air supply fluctuates then most likely your back pressure will fluctuates too and at the same value of DO,% the actual soluble concentration of oxygen in mg/L will be different that you should keep in mind, also when you compare two or more batches you should compare mg/L not DO,%.
One more tip, if your process is going to be scaled-up then no sense to use agitation higher than 400rpm in a lab fermenter because at this rpm you have KLa =300-400 that is typical for large fermenters, if you use higher than 400rpm then you have to re-develop the process in the scale-up fermenter.again that will be very costly.
Hi, I need some clarification.
Let say before calibration the pO2 value in the vessel is around 40% and then we perform calibration. Turn on the aeration and agitation to max to be used.
1. Do we wait until the pO2 reading reached 100% and end calibration
or
2. wait 10-15 minutes, allow the vessel to be saturated and whatever reading (e.g. 60-70%) we adjusted the pO2 value to be 100% and end the calibration.
Please advise.
Many thanks.
Before autoclave, connect DO probe to the cable for at least 6 hrs. Check the response of DO with nitrogen; the response of DO when disconnected the cable. Pre-calibrate the DO at 100%. Check DO slope, raw data to see if everything is in the range.
After autoclave, connect DO with cable to see the response. It should be around 100%
Add media. Start stirrer, heating, air sparge (2- 5% for equilibration). When the temperature is 37C+/-0.C (around 1 - 2 hrs). Calibrate DO at 100%
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