9 replies to this topic

[Biogareth]

Today I did fermentation with XL10gold using double LB medium plus glucose.
I was told it will get high OD, but after incubation 12 hours, the OD is only 7.5, but the pH starts to increase (>7.0).
Could you give me some suggestions? Do I have to stop the fermentation now?
Thanks!

[pDNA]

could you give us some more information on what you are doing?

Regards,
p

[Biogareth]

pDNA, on 09 December 2011 - 01:12 AM, said:

could you give us some more information on what you are doing?

Regards,
p

The purpose for this fermenation is to get more cells, so I tried to use richer medium, but the OD I got is still the same like LB medium.
I just wonder which factor will influence the cell density?

[pDNA]

fermentation in bioreactor or shaker flask?

you should use buffered medium or control the pH ...in batch mode the cells produce a lot of acetate that leads to growth inhibition (crabtree-effect).

If you want to make more biomass in shaker flasks use terrific broth (TB) with glycerol as a carbon source and phosphate buffer.

Regards,
p

[Biogareth]

pDNA, on 09 December 2011 - 05:35 AM, said:

fermentation in bioreactor or shaker flask?

you should use buffered medium or control the pH ...in batch mode the cells produce a lot of acetate that leads to growth inhibition (crabtree-effect).

If you want to make more biomass in shaker flasks use terrific broth (TB) with glycerol as a carbon source and phosphate buffer.

Regards,
p

I used batch fermenation in bioreactor with pH controlled.
One of my collegue used the same rich medium to cultivate BL21 producing another protein, and the OD600 reaches to 25 after 8 hours.
But for my case, only 7.5 of OD was got after 13 hours incubation.
Did you use TB medium to cultivate XL10gold cells before, or e.coli K12? How much OD can be reached?

[pDNA]

i never used complex medium in bioreactor cultivations ...only defined, synthetic medium.
BL21 has a different physiology compared to K12 strains like XL10 ...and you can not assume to reach the same OD with XL10 than with BL21.

E. coli is not E. coli ...the strains differ more than most people believe!

Are you producing a recombinant protein?

I used TB in shaker flasks and as far as i remember i got OD600>10 with JM109.

Regards,
p

[pDNA]

another thing that came to my mind:
The glucose has been added after sterilisation?

Regards,
p

[Biogareth]

pDNA, on 09 December 2011 - 09:12 AM, said:

i never used complex medium in bioreactor cultivations ...only defined, synthetic medium.
BL21 has a different physiology compared to K12 strains like XL10 ...and you can not assume to reach the same OD with XL10 than with BL21.

E. coli is not E. coli ...the strains differ more than most people believe!

Are you producing a recombinant protein?

I used TB in shaker flasks and as far as i remember i got OD600>10 with JM109.

Regards,
p

Thank you pDNA
Yes, I used it for protein production. I also think BL21 can reach much higher OD than XL10gold using same medium.
I did not separate to sterilize with glucose, but BL21 also did the same.

[pDNA]

why you switched from BL21 to XL10?

Regards,
p

[le_duy211]

Could you please tell me the differences between K12 fermentation and BL21 fermentation in bioreactor.