A feeding strategy for tetramethylpyrazine production by Bacillus subtilis based on the stimulating effect of ammonium phosphate.

To examine the effects of ammonium salts on tetramethylpyrazine (TTMP) production by Bacillus subtilis CCTCC M 208157, different ammonium salts were tested, and diammonium phosphate (DAP) was found to have a predominant effect on stimulating TTMP synthesis. The DAP requirements for TTMP production were then investigated, experimental results showed that higher concentrations of DAP favored TTMP production, while both the ammonium and phosphate ions exhibited inhibitory effects on the cell growth and precursor 3-hydroxy-2-butanone accumulation. Based on the results above, a DAP feeding strategy was developed and verified in further experiments. By applying the proposed fed-batch strategy, the maximum TTMP concentrations reached 7.46 and 7.34 g/l in flask and fermenter experiments, increased by 55.1 and 29.0% compared to that of the batch TTMP fermentation, respectively. To our knowledge, these results, i.e., TTMP yields in flask or fermenter fermentations, were new records on TTMP fermentation by B. subtilis.