I am trying to grow up E. coli in a chemically defined media. Currently, I am using a recipe from a paper released by Eppendorf as a guide.
In my recipe, I have
- 100 mL of citric acid/phosphate buffer (133g/L KH2PO4, 40g/L (NH4)2HPO4, 17g/L citric acid) and adjusted to pH 6.7-6.8 using sodium hydroxide (going to adjust citric acid concentration on the next run)
-900 mL DI water
- 11.11mL MgSO4 (240g/L)
-0.25 mL Thiamine (20g/L)
-16.3 mL 70%w/v glycerol
-11.11mL Trace elements (12g/L sodium citrate dihydrate, 0.25g/L cobalt chloride hexahydrate, 0.3 g/L H3BO3, 0.49g/L sodium acetate, 1.07g/L EDTA)
For some reason, all of these items are prepared separately and are perfectly fine in solution. I autoclave the buffer and filter sterilized the rest. and they are all fine. However, once I mix the two, a lot of white precipitate forms. I filtered out the precipitate, and tried to grow bacteria that were once in LB on it (600uL at 0.6 OD) in shake flasks containing 40mL of the filtered media, but their growth is super slow. Can anyone explain what's happening?
You are most likely precipitating magnesium ammonium phosphate, which is not very soluble. The precipitation of phosphate will likely mess up the pH, which would explain why you get poor growth. Growth in mineral medium is slower than in LB medium anyway. Your Mg concentration is very high : 21,5 mM final, whereas most media use 1 mM. So is your PO4 concentration : > 100 mM. Try reducing Mg2+ to 1 mM, or better, use the M9 medium recipe, which you can find on the Internet.
Please look into the
Also refer -
http://www.genaxy.com/uploads/File/catalog.pdf
Does M9 serve as a good media though in large-scale bacterial growth (like 2L in a bioreactor, for example)? If not, how do people usually optimize the media to obtain best productivity?
I agree completely with the Pierre Beguin's answer. Most probably you are getting a big precipitate of Mg phosphate. Final concentration of Mg is very high, and most probably there is a factor of 10 mistake in the recipe.
I agree that you most likely precipitate the phosphate compounds. Moreover, if I compare your medium with a regular medium, your values for MgSO4 and for KH2PO4 are 10 times higher. Double check if you do not have a mistake in the recipe.
Best regards
Geoffrey
I know this post is old, but maybe it can be revived as I also have similar problems. I run fed-batch fermentations in 2 L bioreactors and in such fermentations it is common practice to load a lot of phosphate in the batch medium, and have a lot of magnesium but no phosphate in the feed to keep the two separate. However, there is still magnesium in the batch medium to sustain growth during the batch phase. Often I see cloudyness in the batch medium before inoculation, which dissappears after continued stirring. However, during the long fed-batch fermentations I very often see precipitation. This is difficult to observe, though, as the broth is cloudy from bacteria and everything is being vigorously stirred, but the spargers tend to block, so that I have to move the air inlet filter to my harvest pipe, and sometimes I see a hard cake at the bottom of the fermenter during cleaning (the cake can be dissolved in sulfuric acid without gas formation, if that says anything).
I use the medium described in Korz et al. (1995). pH is adjusted to 6.3 during preparation and adjusted to 6.7 shortly before inoculation.
Shiloach and Fass (2005) seem to have looked up solubilities and conclude that a non-precipitating medium can only sustain biomass to OD = 1. If that is true then many media are oversaturated and only waiting for a chance to precipitate.
pH influence the solubility of Magnesium phosphates as it changes the balance between H2PO4, HPO4, and PO4.
I agree that is true many media are oversaturated and only waiting for a pH chance to precipitate. So, try to maintain the pH of your culture close to 6.7
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