At the end of the glycerol fed batch we obtain a cel concentration between 220 and 280WCW/L. Following this phase, we start with the methanol fed batch and add methanol at a ratio of 3.12mL per hour (start volume fermentor = 2.5L). However, the pO2 drops rapidly and remains below 13% (mixer at max rpm and addition of 40% O2). At the end of our fermentation process, the amount of excreted protein is very low.
Am I wrong to say that the amount of MeOH we are adding is very low and still we are not able to maintain the pO2 at 20%. What is going wrong here?
For your information, due to pump restraints we cannot add MeOH at a speed below 3.12mL an hour.
Dear Veerle,
I am not a P. pastoris expert but I find your question intriguing
Do you use a mutS strain of P. pastoris - and have you confirmed that methanol is completely oxidized to CO2? Methanol uptake capacities of mutS strains are really low. Perhaps this thesis, that I came across via Google, is relevant to check if you are in a safe region (for comparison with the data in the thesis, it would then be helpful to have biomass dry weight measurements, so that you can calculate biomass specific methanol uptake rates).
http://herkules.oulu.fi/isbn9789514295850/isbn9789514295850.pdf
I am a bit surprised that this very low rate of methanol feeding has such a huge impact on oxygen uptake - assuming that you use a high performance bioreactor. This might be (but is not necessarily) an indication that formaldehyde and/or formate are accumulating.
Good luck
Jack
With this very low feed rate, can you prevent dropwise (feast/famine) additions?
Hi Veerle
The MutS Strains have a reduced Methanol consumption and your value should be in the described range, but there are lower descriptions for used optimum levels, like 0,66ml/h in this papers:
http://www.pp.bme.hu/ch/article/viewFile/253/147
or discontinous here:
http://www.microbialcellfactories.com/content/11/1/22
You might not be able to undergo your pump minimum, but you can dilute Methanol in water to reach lower levels. The dropping of your pO2 can be a physical effect of substituiting oxygen with Methanol steam, the MutS does not metabolize it so very fast so you will not have a chance to change this. Maybe you just have an empty exhaust of unused Methanol in the outgoing air stream, I would think that you might benefit in really reduce you Methanol input, for inducing the expression, lower levels should fit and your Strain does not have to get rid of exceeding Methanol-Level and might express easier and better.
Do you monitor your level of formic aldehyd and formic acid?
good luck
Dear Jack and Frank,
Thank you for your answers!
The thing is that we work with a Mut+ strain, so the metabolism of MeOH should be alot higher than what we observe...
What we saw in our latest run, was a pO2 very rapidly fluctuating between 8 and 31%. Could that be caused by the MeOH steam (we use the Oxyferm FDA 325 probe from Hamilton)?
Frank, we don't monitor the level of formic aldehyd and formic acid. How do you perform this monitoring?
Thank you in advance!
Hi Veerle
In would suggest that your pO2 "problems" are influenced by the measurement....it is to quick to be metabolic and small volatile molecules can easyly influence the membran transfer of oxygen. We usually monitor many metabolic systems vie HPLC, you have a few possibilities for organic acids like broad band Ion exchange columns with refractrometric detection, the formic aldehyd is monitored their too, you have a possibilities to measure photometrically with quant. wet chemical Aldehyde Kits, or with Purpald, if you dont add other carbon source you might quantify formic acid via increase of total acids, there are a few photometric or reflectometric kits avaialable.
Sometimes I us a Merck Reflectoquant Pocketlab, a small mobile unit that offers a good selection of wet chemical tests.
I think you might monitor the metabolism via estimating the formic acid and aldehyd too.
Good luck
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