Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs

Abstract

Bacillus subtilis spore preparations are promising probiotics and biocontrol agents, which can be used in plants, animals, and humans. The aim of this work was to optimize the nutritional conditions using a statistical approach for the production of B. subtilis (WHK-Z12) spores. Our preliminary experiments show that corn starch, corn flour, and wheat bran were the best carbon sources. Using Plackett-Burman design, corn steep liquor, soybean flour, and yeast extract were found to be the best nitrogen source ingredients for enhancing spore production and were studied for further optimization using central composite design. The key medium components in our optimization medium were 16.18 g/l of corn steep liquor, 17.53 g/l of soybean flour, and 8.14 g/l of yeast extract. The improved medium produced spores as high as 1.52 +/- 0.06 x 10(10) spores/ml under flask cultivation conditions, and 1.56 +/- 0.07 x 10(10) spores/ml could be achieved in a 30-l fermenter after 40 h of cultivation. To the best of our knowledge, these results compared favorably to the documented spore yields produced by B. subtilis strains.

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production

“…These values do not surpass the spore cell density reached by Chen et al [9], who reported a concentration of 1.52 9 10 10 CFU/mL for the B. subtilis WHK-Z12 strain, but to our knowledge, the sporulation efficiency reached in this work (93.5 ± 4.0 %) is the highest reported to date for B. subtilis strains that are in the range of 30-80 % [11,12,14].…”

“…Furthermore, during the stationary growth phase in a fermentation process, when nutrients are exhausted, the culture initiates sporulation at a cell density of about 10 8 cells/mL [8]. Under ideal conditions, typical cell densities and sporulation efficiencies for B. subtilis will be in the range of 1.00 9 10 8 -1.52 9 10 10 CFU/mL [9][10][11][12] and Electronic supplementary material The online version of this article (doi:10.1007/s00449-015-1428-1) contains supplementary material, which is available to authorized users. 30-80 %, respectively using serial dilution and plate count methods [11][12][13][14].…”

“…Elements such as Ca, Mn, Mg, Fe and Zn in appropriate concentrations are essential in sporulation since they are present in the spore layers and enable it to resist high temperatures [11] and the last two elements accelerate the sporulation process [3,20]. Regarding the culture conditions, several studies have evaluated the effect of different effective volumes (1-70 L), aeration rates (0.5-2.0 vvm), agitation rates (200-500 rpm), pH (5.0-9.0) and inoculum size (1-3 %) in order to obtain large amounts of biomass and secondary metabolites [3,9,12,21,22], concluding that optimum culture conditions are very specific for each strain. Therefore, optimization of culture medium and culture conditions of new strains can lead to industrially viable processes.…”

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

“…The production of the phytohormone (IAA) and siderophores by Bacillus species has been investigated in many studies. The IAA stimulates the plant growth and siderophores chelate iron (Fe) and deprive the phytopathogenic fungi of it [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Here, the Bacillus spp.…”

“…Spores concentration in Bacillus cultures was investigated using a thermal chock technique [8]. All experiments were performed as three replicates.…”