I grow Rhodococcus in shake flasks with synthetic medium containing 1.0g NH4NO3, 0.2g MgSO4-7H2O, 1.0g K2HPO4, 0.15gKCL, 0.1g yeast extract and a trace element solution. Different alkanes are added as the only carbon source. The pH changes from 7.0 to 3.9 over night, but I need to extend cultivation time for better hydrocarbon degradation. What is a good way to buffer the medium at 7.0 against this large pH change without changing the nutrient content of the medium?
Thanks
try buffers such as MOPS, HEPES or TRIS, with pKa values ideal for your operational pH (7.0). as for the concentration, I would recommend you to start low with a 20-30mM and further increase until you reach your desired pH.
Hope this helps
It contains quite little phosphate compared with other synthetic media which usually use 50 mM of phosphate and you barely have 5.7 mM, almost 10 times less.
The question is not only the change in nutrients but also that you will be changing the osmolarity of the medium. Keep that in mind.
Thanks Xabier, so do you recommend buffering with K2HPO4 and KH2PO4?
Usually, you use hydrogenphosphate and dihydrogenphosphate because depending on the ratios you mix them you can get the pH you could even use plain phosphoric acid and adjust the pH, in the end the speciation of the Pi is going to be the same, but the buffering capacity of your medium will depend on how much Pi you have.
By the way, where do you get that recipe from? It kinda looks relatively hypotonic compared to similar minimal media. In my former lab, we used the medium described in the paper linked for the degradation of PAH and fuel, it is very similar to the BH medium
Article Isolation of novel benzo[a]anthracene-degrading microorganis...
Thanks so much for the info! Its from the paper that originally isolated the organism I am working with and has been used in all the subsequent papers they published on this bacterium.
I use MES at 10mM for a defined media for a clostridia that can induce a shift from 6.5 to 4.0 over 24 hours.
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