Production of tetramethylpyrazine by batch culture of Bacillus subtilis with optimal pH control strategy

The effects of initial culture pH ranging from 5.0 to 7.5 on biomass content, precursor 3-hydroxy-2-butanone (HB) accumulation, and 2,3,5,6-tetramethylpyrazine (TTMP) formation by Bacillus subtilis CCTCC M 208157 were investigated in shake flask fermentation. Weak acidic conditions were found to favor cell growth and precursor HB accumulation, while TTMP could be synthesized more efficiently in conditions with initial pH towards neutrality. Batch bioprocess of TTMP fermentation by Bacillus subtilis CCTCC M 208157 at various controlled pH values ranging from 5.5 to 7.0 was then examined in 7.5-l fermentor. The results suggested that optimum pH for cell growth and precursor HB accumulation was 5.5 with maximum cell growth rate (Q (x)) and precursor HB accumulation rate (Q (HB)) of 0.833 g l(-1) h(-1) and 1.118 g l(-1) h(-1), respectively, while optimum pH for TTMP formation was 7.0 with maximum TTMP formation rate (Q (TTMP)) of 0.095 g l(-1) h(-1). A pH-shifted strategy was accordingly developed to improve TTMP production in bioreactor fermentation by shifting the culture pH from 5.5 to 7.0 after 48 h of cultivation. By applying the strategy, final TTMP concentration of 7.43 g l(-1) was obtained, being 22.2% greater than that of constant-pH fermentation.