Hi,
I am working on Pichia pastoris fermentation. I had runned on 7.5 litter scale, small scale. I have yet tried at 25 litter scale, big scale.
The result shows I got failure in scaleup.
You can see in my attached file. The big fermentation has yeast that grow slowlier and lowlier comparision with the small one. It takes 40 hours to reach induction time, longer than the small. Despite DO in the big was always been kept above 100% because of effective system, its protein expression profile clearly worse than the other.
I repeated in 3 times and got the same result.
I fell into despair because I can not figure out what is going on.
Does somebody else had been in my situation? I am looking for your advice.
Thanks.
Hello, it can happened the changing volume twice and starter twice dose not mean you should receive identical growth patterns. It not chemistry it's living organism and many factors can influence.
What is the volume of the seed culture in 25 L bioreactor? Secondly it shall be from an early or mid-log phase seed culture. The dO2 seems static and there is no improvement in the wet cell mass, too.
Shamsher S. Kanwar the seed volume of 25L fermentation is 1.5L BMGY. An additional information, before the induction by methanol, yeast grew in BSM medium with 4% glycerol. therefore you can see a secondary log phase after induction. It means yeast was growing in methanol
Hello, it can happened the changing volume twice and starter twice dose not mean you should receive identical growth patterns. It not chemistry it's living organism and many factors can influence.
Karen A. Darbinyan
I do not understand clearly. I know the final iead that it can be afffected by many factors. And I am figuring out what is matter.
Could you explain in more details?
Dear Trung Nghia,
Your results confirm that the hydrostatic pressure in the fermentation mass negatively affects the metabolism of the fermentation microbiota. I have carried out some experimental studies on this subject in pressure ranges of 0-6 bar and I noticed a reduction of microbiological activity up to 10 times under the conditions of maximum pressure tested. Although the pressure difference in your experiment is much lower, this is obviously an inhibitory factor that prolongs the lag phase and reduces the fermentation yield. The fermentation vessel geometry (vertical or horizontal) influences the more or less pronounced inhibitory effect.
Hello. Please do not despair, Scale up is not easy.
It is difficult to figure out exactly what has gone wrong from the information that you have provided, but I shall make some suggestions. And I am sorry if these questions are too simple, I do not know how much experience you have.
The good thing here is that your strain did not die at inoculation in the larger vessel. The lag phase is obviously much longer than expected.
I have noticed that if you induce a tank early (= at a relatively low wet cell weight), then the product yield will decrease. You have noticed that too with your system.
The DO curve of the small vessel worries me a bit.
Do you know why the DO increased from 12 to 16 hours?
Were you adding a carbon source?
Were you increasing the agitation speed?
Were you increasing the gasflow rate?
Were you increasing the pressure?
The DO curve of the large vessel worries me a bit more.
If I see a constant reading, then the first thing I would check is the probe itself.
If the DO probe has a membrane:
Did you change the electrolyte (no bubbles) before each experiment?
Was the membrane intact when you removed it at the end of the experiment?
Was the cable connected properly?
Was the DO probe calibrated properly?
If the DO probe is an optical probe:
Was the cable connected properly?
Was the DO probe calibrated properly?
If you are able to provide more information about your experiments, then my follow up suggestions might be a bit more useful for you. But I do understand that sometimes you are not able to provide much detail in a public forum.
Keep trying.
Deborah Mun
Thanks for your response. I'll answer one by one of your questions to give you a clearly view about my case.
- Do you know why the DO increased from 12 to 16 hours?
>>> It causes by using pure oxygen. At the first 12 hours, I used normal air to support oxygen, but you see, it isn't enough for yeast and I change to the other source.
- Were you adding a carbon source?
- Were you increasing the agitation speed?
- Were you increasing the gasflow rate?
- Were you increasing the pressure?
>>> At this time, I did not add the carbon source. It happened at 20th hour. The agitation speed, pressure had been kept at normal. The flowrate decrease clearly because of changing oxygen source.
- The DO curve of the large vessel worries me a bit more.
>>> Actually, the value of DO is higher than 100%, but the logging software transform all values uper 100% to 100%. I want to say that DO of the large bioreactor had always kept above 100% because I used pure oxygen and an effective air sparger. By the way, I define 100% of DO is the maximum dissolving capacity of oxygen from air to the fresh medium at initial time. Therefore, when I change to pure oxygen, the DO value may be larger than 100%.
Hello Trung, I have recently encountered this problem in my biosurfactant and L-asparaginase studies. I assure you that I can be frustrating. What I did was to refer to review article on Scale-up of industrial microbial processes by Crater and Lievense, 2018. There are eight parameters that dutifully influence fermentation scale-up performance. If you can painstakingly identify sort them out, you will be a happier man. I have attached the document. Just go through it. I hope it helps because it did help me.
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