We do G-CSF fermentation using recombinant E.coli cells. After inoculating into the fermenter following a successful media hold, Dissolved oxygen % is dropping to zero within three hours. Oxygen even if sent at maximum, DO is not rsing more than 2%. What can be the reasons??
Dear Vishnu,
how strong do you inoculate (initial optical density)? High cell densities under producing conditions need a very O2 supply.
With pure oxygen at higher aeration rate, you should see at least an effect on DO.
Did you check a different oxygen probe? How was the sensor calibrated? Effect could come from a defective or wrong calibrated electrode.
Best, Michael
Hi Michael,
Yes, We have changed the whole set of DO probe and the transmitter. Two point calibration was done as per procedure. But nothing seems to work. Let me know if there are any suggestions.
Hi Vishnu
As the colleagues mentioned before: your O2 consumption is too high...
Can you show us the optical density, volume, the aeration rate, the gas and media that you use?
Aeration is a critical parameter for many scale-ups, some systems offer automated regulation within a certain frame by increasing of stirring, increasing of aeration and increasing the back-pressure of the system.
You might increase the foam with higher aeration too, but some anti-foams (pluronics f.e.) might help you increasing the dissolved O2 rate.
By the way 2% is better than nothing, if you have a high cell density or a log growth, you need to change a few things to avoid the oxygen limitation and reach an above-zero-state.
Perhaps your carbon source can be reduced, feeded or the feed-rate can be lowered if you have no other technical possibilities.
Hi guys,
We have tested every possibility. A 100% manual increase of oxygen is not hiking the DO. Not even a spike of DO is observed in the trend. There is no back pressure maintained usually. Increasing the back pressure hikes DO to 2% only. What can be the reason?
Oxygen uptake rate depend on biomass growth rate. At the start of the fermentation the biomass growth rate may be extremely high. That is the reason of dropping DO to zero. Later during the fermentation the biomass growth rate decreases and DO increases. But during some period of fermentation DO is zero or something like that. Such a situation is not good for the processes, where the goal is not biomass but some methabolite product. In my patent (USSR, 30 years ago) we proposed during this "hot period" to decrease the temperature in order to maintain DO on the optimal level. After finishing this "hot period" we return the temperature to normal level. I suppose that such a procedure will be good in your case.
Agree with Prof Biryukov that it sounds like you will need to adjust conditions (temperature is a good one) to suit your particular needs in the process. Good luck.
Thank you for the suggestions. But this process is not a new one. We have been doing the same procedure since 6 years successfully. But all of a sudden, batches are behaving this way. Please let me know what I'm missing here.
Well, clearly there is oxygen limitation. Not unlikely for exponential growth after some time. When this did not happen before, look at a reason for the change: either a larger consumption or a smaller (measure transfer rate using offgas concentration using a temporarily stop in aeration; indeed enrichment of the gas used will increase mass transfer and a conclusion if either OUR or OTR is the reason. When OUR check e.g. for infection. When OTR check for changes in your broth, like use of antifoam.
Success.
Hello Vishnu: according to my opinion the best way to solve your problem is as follow:
i) determine oxygen transfer rate in your fermenter under different aeration rates and rpms
ii) measure oxygen consumption rate of your bacteria
iii) compare both values: if oxygen consumption rate is > oxygen transfer rate, everything is clear, if oxygen consumption rate is < oxygen transfer rate, then a miracle is going on in your fermenter!! :-))!!
Best regards
Vit