In the literature review, it is written that precipitate of PHB is obtained. But I am getting no precipitate even after centrifugation.
Maybe the bacteria doesn't have a polymer. You need a more detailed extraction Protocol that you used.
Thank you Evgeniy Kiselev for your reply. The presence of of PHB was confirmed by Sudan Black B in the agar culture of same bacteria. Could you please recommend or provide me a detailed protocol?
Classically, chloroform is used for extraction, but the yield may be low due to the strength of the cell wall. Also, the biomass moisture will affect the extraction efficiency, since water does not mix with chloroform, therefore it will prevent the penetration of the solvent into the cell.
Therefore, the laboratories often use the Folch method, which implies the use of a mixture of chloroform: methyl alcohol in a 2: 1 ratio.
The second important aspect is the solubility of the polymer in a solvent, usually it does not exceed 6% (but it all depends on the molecular weight of the polymer). On this basis, the recommended ratio of biomass: solvent is at least 1:10, and preferably 1:12 or 1:14.
Third. The specifics of polymer dissolution should be taken into account, and this process is not fast. First, the solvent is migrated to the polymer matrix, then the polymer swells, and only then the polymer begins to go into solution. And in your case, the obstacle is still the cell wall and the water that is contained in the biomass. Therefore, I recommend a duration of 12 to 24 hours.
Well, for the precipitation, you can use one of the following solvents: hexane, methyl alcohol, ethyl alcohol, isopropanol, acetone. The main ratio of extract: precipitant, which should be 1: 4.
As far as I remember, Sudanese black is not a selective dye for PHA, but colors the entire lipid fraction, but I can be wrong. Check it out in the literature.
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