I have Pichia pastoris X-33 with pPICZalpha with my GOI.
It's grown in bioreactor Biostat Cplus (Sartorius) in 2x YNB, 100 mM K-Pi,4% glycerol @28°C.
I put the inoculum in on Thursday, since the evening went down, in the morning I increased rpm to 1000 and the oxygen continued to go down (we have no oxygen to supply, only air, which is at 2 vvm). In the other bioreactor we had, it usually take Pichia the whole weekend to use all the glycerol, so I were not sure what to expect, but based on some articles, I supplemented them with 1l of 50% glycerol over night (so about 10 ml/l/h or less). The oxygen continued to decrease until about 4AM today when after short plateau it started rising. At the same time, the pH started to rise as well (we have always used only KOH for pH control, as the acid was never neccessary). In the morning, the pO2 was at ~18 and pH at 7.3 or so (starting at 7.0), I loaded the rest of the glycerol from the night and the oxygen dropped immediately to ~13 (really like in 5 minutes) and pH also somewhat went down (the glycerol contains PTM1 which are acidic). However, since that time I'm loading another 50% glycerol at the rate 20ml/l/h (as I've seen in few articles), but the oxygen level goes up, instead of decreasing. And the pH is at the same level (althought the acidic PTM1 are added). I measured the OD600 in the morning it was ~463, now, after 3.5 hours it was ~536, so it seems it's alive.
So the questions are
1) why would the oxygen drop so quickly just because I increased the speed of glycerol loading? The yeast could hardly respond so quickly so I don't think it has something to do with them.
2) why would the pO2 and pH go up (or stay steady in case of pH, when the minerals are added), when the yeasts should consume them.
3) is there any reason why should the yeast increase pH, except of cell lysis? I hope the cells are still viable even if they were hungry for one night.
Just an update. I attached an image of the process. In red is the pO2 with the main points marked. As you can see, during this night, while feeding with MeOH at 75 ml/min, the pO2 started to decreased, at first slowly, then rapidly and then increasing again. I decreased the MeOH feed rate, because I was afraid I would kill the yeast if they do not consume it, there was pumped during the night about 1200 ml into volume of ~12l.
BTW the OD600 is still somewhat increasing. While in the evening it was ~570, in the morning it was ~660.
Hi Tomáš
The pPICZ alpha has a Zeocin Selection Marker, did you use this in your preculture?
I would propose this for minimum for the precultures, and the working cellbench, because of the price you can leave it out in the main culture
Do you start from a propper Startingculture or do you use an inoculum from a plated clone? This is a proposed procedure from books like Maniatis, to use somewhat "fresh" plated cells,practically a rich starter culture, frozen in good condition and big celldensity in a log growth is far more reproducible if you really want to do a fermentation....
Hi,
thanks for your answer.
No, I didn'z use Zeo in preculture. I used selection on methanol :) But the culture seems OK in terms of contamination.
This time I grew it from some leftovers in the fridge by which I've inoculated 5 ml and from that the other day 500 ml which I used for inoculation of the fermentor.
However, right now I'm more interested why would the oxygen behave so weirdly? If the yeast have just enough of methanol, they should consume the oxygen, right? So when I feed them with MeOH the pO2 should go down, right?
To exclude any kind of disturbance from unknown growth: did you check for contaminations in plate ot microscope?
The other proposal would be a inconsistence in you working cell bench, store the vials from a Zeocin growing culture to exclude´wrong positives
Maybe a change of the main culture medium can help. YNB is not cheap, you have other, more rich production media that are less expensive, if you focus on a technical scale fermentation YNB would not be the choice anyway
I found one paper with a similar focus, perhaps this might give more hinds.
Yes, I had a look under microscope and it seemed OK (although I have no idea what to look for, but at least there were no bacteria).
It seems I simply put too much methanol into the fermentor. Yesterday was the oxygen slowly decreasing with up to ~5ml/l/hr. Unfortunately there was a power shut-down in the evening so the stirring turned off.
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