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

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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 \( 1.52 \pm 0.06 \times {10^{10}}{\text{spores}}/{\text{ml}} \) under flask cultivation conditions, and \( 1.56 \pm 0.07 \times {10^{10}}{\text{spores}}/{\text{ml}} \) 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.

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Acknowledgments

We thank Dr. Jian-Ping Liu (Max Planck Institute for Infection Biology, Berlin, Germany) and Dr. Barry Wong (Wuhan University, P. R. China) for their advice and critical reading of the manuscript. This work was funded by the National Natural Sciences Foundation of China (NSFC Grants No.30770042).

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Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People’s Republic of China

    Zhen-Min Chen, Ming-Yuan Yang, Ping Shen & Xiang-Dong Chen

  2. Wuhan Kernel Bio-Tech Co., Ltd, Enterprise Technology Center of State, Wuhan, 430074, People’s Republic of China

    Qing Li, Hua-Mei Liu, Na Yu & Tian-Jian Xie

Authors
  1. Zhen-Min Chen
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  2. Qing Li
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  3. Hua-Mei Liu
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  4. Na Yu
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  5. Tian-Jian Xie
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  6. Ming-Yuan Yang
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  7. Ping Shen
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  8. Xiang-Dong Chen
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Corresponding author

Correspondence to Xiang-Dong Chen.

Electronic supplementary materials

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Table S1

Independent variables studied in Plackett–Burman screening design (DOC 35 kb)

Table S2

Plackett–Burman design for the ten independent variables in coded values and measured responses (DOC 55.5 kb)

Table S3

Regression analysis of Plackett–Burman design for spore production (DOC 33 kb)

Table S4

The design and results of the path of steepest ascent experiment (DOC 29 kb)

Table S5

Coded and actual values for the independent variables in the central composite design (DOC 30 kb)

Table S6

Optimization of spore yields and experimental results for central composite design (CCD; DOC 40 kb)

Table S7

Regression analysis of central composite design in B. subtilis (WHK-Z12)spore production (DOC 33 kb)

Table S8

Analysis of variance for the model fitting spore yields (DOC 31 kb)

Table S9

Ridge analysis for the maximal spore yields of B. subtilis WHK-Z12 (DOC 40 kb)

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Chen, ZM., Li, Q., Liu, HM. et al. Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs. Appl Microbiol Biotechnol 85, 1353–1360 (2010). https://doi.org/10.1007/s00253-009-2162-x

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  • DOI: https://doi.org/10.1007/s00253-009-2162-x

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