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Rapid Optimization of Spore Production From Bacillus Amyloliquefaciens in Submerged Cultures Based on Dipicolinic Acid Fluorimetry Assay


Rapid Optimization of Spore Production From Bacillus Amyloliquefaciens in Submerged Cultures Based on Dipicolinic Acid Fluorimetry Assay

Hang Ren et al. AMB Express.


Some optimization techniques have been widely applied for spore fermentation based on the plate counting. This study optimized the culture medium for the spore production of Bacillus amyloliquefaciens BS-20 and investigated the feasibility of using a dipicolonic acid (DPA) fluorimetry assay as a simpler alternative to plate counting for evaluating spore yields. Through the single-factor experiment, the metal ions and agro-industrial raw materials that significantly enhanced spore production were determined. After conducting a response surface methodology (RSM) analysis of several metal ions, the combined use of optimum concentrations of Mn2+, Fe2+, and Ca2+ in culture media produced a 3.4-fold increase in spore yields. Subsequently, supplementing soybean meal and corn meal with optimum concentrations determined by another RSM analysis produced an 8.8-fold increase. The final spore concentration from a culture medium incorporating optimum concentrations of the metal ions and raw materials mentioned above was verified to reach (8.05 ± 0.70) × 109 CFU/mL by both DPA fluorimetry and plate counting. The results suggest that the use of DPA fluorescence intensity as an alternative value to colony counting provides a general method for assessing spore yields with less work and shorter time.

Keywords: Bacillus amyloliquefaciens; Dipicolinic acid; Fluorescence intensity; Response surface methodology; Spore yields.


Fig. 1
Fig. 1
Calibration curves of spore counts of B. amyloliquefaciens BS-20 and their fluorescence intensity. The spores of B. amyloliquefaciens BS-20 with 1.34 × 108 CFU/mL were twofold serially diluted and treated for the detection of fluorescence intensity
Fig. 2
Fig. 2
Response surface plots for spore production caused by metal ions. The interaction between a Mn2+ and Fe2+, b Fe2+ and Ca2+, c Mn2+ and Ca2+, respectively
Fig. 3
Fig. 3
Response surface plots for spore production caused by soybean meal and corn meal

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