Hello everybody,
I'm currently growing E. coli in bioreactor and I have experience only with P. pastoris in bioreactor, so I'm not sure, how should it behave. With Pichia, one can try hard to imrove the conditions and it will grow very well to very high cell density (something like OD600 of hundreds), so I had similar approach with E. coli.
I usually grow E. coli (in flasks) in LB medium supplemented with 0.1M K-phosphate pH 7.0; 1% glycerol; 2mM magnesium.
I grew it for the first time in this medium, but the pH shifted up to 8.x after switching the temperature to 18°C (after induction). We thought it was because of the phosphate, although from what I found on the internet, the phosphate buffer shall not be so much sensitive to temp change as other buffers. Anyway, we grew it without the phosphate the second time, but the pH increased anyway (not that much though). Moreover, it was slowly increasing over time, rather than decreasing. It was about 7.2 in the morning (maybe more). What could be the cause of the pH increase?
Second- what is the usual OD600 after overnight growth in bioreactor? The first time we got around 8, but the pH was slowly increasing. At that time I thought it's because of cell lysis, but now in retrospect, it could be related to the changes in pH as mentioned above. The second time we got OD600 around 3, but it was after shorter time (because we thought it's lysing) and the culture grew slower in general in comparison with the first one.
Thank you for your suggestions.
Hi,
Some comments about what you describe above:
1) The pH increase in your described culture is expected, and is observed when growing E. coli in complex media containing amino acid sources (such as LB). As the cells grow they will consume amino acids from the medium, resulting in the removal of acid equivalents and consequently an increase in medium pH. If you instead use defined media without amino acids, the bacteria will instead consume a nitrogen source, often NH4+, from the medium. This metabolism results in a net release of protons and, together with acetate production, leads to a lowered pH in the medium.
2) Your described medium will not support growth to ODs in the hundreds, as there is not enough available nutrients for this. 10 g/L of glycerol will give you roughly 5 g dry cells/L, for an OD in the range 10-15. Add in another 10-15 from the available carbon in the complex components, and you are looking at maximum 20-30, but I would assume some other component (or oxygen!) will become limiting before you reach this high. My suggestion would be to switch to a defined salts medium suitable for high cell density culture and performing fed-batch culture if you need to reach ODs in the hundreds.
Some useful papers:
Article Microbial growth and physiology: A call for better craftsmanship
Article High cell-density culture of Escherichia coli
Dear Martin,
thanks for your fast response.
Of course I didn't mean that E. coli would grow to hundreds (is that even possible?). I meant that Pichia is able to grow that much.
Anyway, the LB medium shall acidify during growth because of excreted acids, shouldn't it? That's what we were always taught. And presumably they observed that previously when they used simple LB medium.
Thank you for the articles, I'll try to go through them.
Intuitively, you would think that E. coli would acidify the medium by excreting acids. However, you should keep two things in mind:
1) Aerobically, E coli makes acetate due to overflow metabolism. This does not occur to a significant degree from glycerol, however.
2) During anaerobic/oxygen-limited conditions, various acids are excreted. This is likely what you have previously seen and learned from flask cultures in LB. These tend to rapidly become oxygen limited due to the fast growth in this rich medium. In a bioreactor you have a higher oxygen transfer and can avoid this becoming a factor until higher ODs, and the dominating effect becomes pH increase due to amino acid consumption (when amino acids are used as carbon source, basic ammonia is released as a side product).
OK, that makes sense. I must admit I also weren't quite sure about requirement of E. coli for oxygen. But it doesn't seem to be limiting factor, as the pO2 decreased only from ~20 % to ~15 %.
However, the main concern is currently about the sudden change of pH upon temperature change. What could that be?
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