[petermakram]

Hi, my name is Peter, I am a research pharmacist working in the development of bio pharmaceuticals. I have some questions about the upstream process:

How should i calculate the volume of the inoculum (pre-culture), based on initial media volume only or initial media volume plus volume of supplments?
What is the accurate equation that I should use to calculate the accurate volume of the inoculum to inoculate the main fermentation?
How should i calculate the feed rate, based on initial media volume only or initial media volume plus supplements plus inoculum?
What is the protocol of measuring the cell density using spectrophotometer, should I use WFI or blank media as a reference while measuring?

If anyone knows a good forum or site that can explain all these fermentation basics ?
Thanks in advance for your answer

[SunilNagpal]

Hi Peter,

You'll need to perform culture optimization trial experiments on a small scale to ascertain the ideal OD to initiate a culture. Once that OD is ascertained, deciding the inoculum size/volume for larger scale can easily be done using:
ODI x VI = ODO x V2
where ODI is the OD of your inoculum stock
VI is the volume of inoculum stock required
ODO is the optimal OD for initiating the culture (deduced using trial experiments)
V2 is the volume of the media or broth.

Feed rate should always be based on total volume.

You should use blank media as reference.

PS: By culture optimization, I mean that you should find out the ideal OD of culture broth (for the specific bacterial strain) that leads to a good growth curve (minimal lag phase). To avoid multiple trials, you may take the OD at the inflection point of lag and log phase, as the optimal OD for initiating the culture.

Hope it helps

Best wishes
Sunil

[petermakram]

Thanks a lot Sunil for your reply.