I would like to harvest Hansenula polymorpha [methylotropic yeast] culture using centrifugation. The optical density of the culture reaches 300 at 600nm [OD600 reaches 300 -350]. What are the optimum centrifuge parameters for such culture cell clarification in terms of RCF, time and temperature?
Hi Peter,
Since these cells are bigger, they settle down very easily, even if you leave them without shaking. I would say you can proceed with 1500-3000g for 5-15 minutes at 4 degrees. This also depends on the volume you want to spin. Therefore you can increase or decrease the time accordingly.
Keep in mind that if you are preparing electrocompetent cells, then start with the minimum speed and time and also keep everything at 4 degrees.
For or after expression you can increase the speed and reduce the time, if you want it done quickly.
Hi Lav,
Thank you very much for your answer.
I would like to add that my aim from this centrifugation is to clarify the cells and collect the supernatant for recombinant protein recovery. I usually perform the centrifugation for 45 ml of culture broth in a 50 ml falcon tube with 3125g for 10 minutes at 4 degrees but I end up with having a loose cell pellet (not rigid) knowing that the optical density at end of fermentation ranges between 300 to 350, which makes the sterile filtration step of the supernatant very difficult.
Why don't I get a rigid cell pellet at end of centrifugation and a clear supernatant? Shall I change any of the centrifugation parameters to have a rigid pellet?
Thanks & best regards,
Hi Peter,
Good that your recombinant protein is in supernatant, this makes your life easy. If you do not need the pellets for future purpose, then you can increase the speed of your spin. Generally, it is observed that you obtain loose pellet with lower spin speed. You can easily go upto 10000 to 15000 RPM for 15-30 minutes@ 4 degrees. Since, the protein is soluble and present in supernatant, it will not come in pellet.
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