Abstract
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.
Highlights
Bacillus species are aerobic or facultative anaerobic, sporulating, rod-shaped bacteria (Driks 2002)
dipicolonic acid (DPA) fluorimetry assay for quantifying the spore concentration Figure 1 shows the good linear correlation between the spore concentrations varying from 8 × 103 to 8 × 106 colony-forming units (CFU)/mL, and corresponding DPA fluorescence intensity
Several studies have been performed on the enhancement of spore production, and the top 2 highest documented spore concentrations of Bacillus undergoing
Summary
Bacillus species are aerobic or facultative anaerobic, sporulating, rod-shaped bacteria (Driks 2002). They can form protective endospores that allow them to tolerate harsh environmental stress, such as heat, radiation, desiccation, freezing and chemical disinfectants (Setlow 2006). The Bacillus spores can survive through the digestive process and germinate within the digestive tract (Casula and Cutting 2002). Endospores of Bacillus are formed after the exponential phase of vegetative cell growth as a result of nutrient depletion and cell accumulation (Driks 2002). In the commercialization of Bacillus-based bio-products, high spore yields in bioreaction with less cost are preferred in industrial exploitation (Chen et al 2010; Khardziani et al 2017; Lalloo et al 2009; Posada-Uribe et al 2015). The regulation of sporulation parameters in fermentation was often carefully considered for enhanced spore production (Monteiro et al 2005; Rao et al 2007)
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