I use LB media supplemented with cutin, but the pH of the media goes beyond 12 which creates problems for P-nitrophenyl butyrate assay, since pNPB is not stable under alkaline pH conditions (> pH 8). Any ideas regarding how to control increased pH or alternative media with buffering conditions would be helpful.
Does your medium has KH2PO4 / K2HPO4? if so it might help to maintain the pH (this is for shake flask, where you have no pH control). If you are using fermentor, you can control the pH with 33% H2SO4 and 6 M NaOH , such high concentration of acid and base will avoid volume increase too.
My medium doesnot have KH2PO4 or K2HPO4.. preparing media in buffers like Na-Phosphate buffers will help pH control??
The pH of the LB is close to Neutral range. I think you need to pretreat your cutin before addiing it to LB. It appears from your question that cutin that you are adding in LB may be alkali extracted and its not properly washed to bring the pH to neutral range. Try to resuspend the cutin in water and then check the pH . I am pretty much sure that your cutin suspension in water will show alkaline pH.
Another possibility is that your strain is an alkalophilic strain so the pH of the medium is shooting to such value of 12 and may be secreting alkaline cutinase enzyme. In that case the organism will readily grow at alkaline pH. However when you have to do or perform the assay you can bring down the pH of the supernatant to neutral by addition of acid so that p-niteophenyl assay at 405-420 nm can be performed. Hope I have been able to answer your query
Use of Na-Phosphate may help, but most people use K-phosphate salts to control pH.
I agree with Deepti regarding thorough washing of cutin before adding to the medium.
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