Enhanced Spore Production of Bacillus subtilis Grown in a Chemically Defined Medium


Spores of Bacillus subtilis are being used as probiotics and competitive exclusion agents for animal consumption. Commercial production media often include relatively expensive components of animal origin that are a potential source for the presence of adventious agents, therefore undesirable for use in production scale. In this study a new animal-free component, chemically defined medium, was tested for B. subtilis spore production. Medium composition was optimized with respect to vitamin composition, carbon, nitrogen and calcium concentrations. A fed-batch bioprocess was developed, being the effect on sporulation of the carbon to nitrogen ratio at the end of the exponential growth phase studied. The developed strategy consisted of an initial and a final batch phase and an intermediate fed-batch phase with the addition of a feeding solution containing glucose and calcium and the addition of a feeding solution of ammonium sulphate, using an exponential and a constant feeding profile, respectively. Using the fed-batch strategy, it was possible to achieve a maximum spore production of 3.6 × 1010 spores/mL, corresponding to a 5 folds increase when compared to the preliminary batch experiments.

Share and Cite:

Monteiro, S. , Clemente, J. , Carrondo, M. and Cunha, A. (2014) Enhanced Spore Production of Bacillus subtilis Grown in a Chemically Defined Medium. Advances in Microbiology, 4, 444-454. doi: 10.4236/aim.2014.48049.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Oliveira, J.E., Van der Hoeven-Hangoor, E., Van der Linde, I.B., Montijn, R.C. and Van der Vossen, J.M. (2014) In Ovo Inoculation of Chicken Embryos with Probiotic Bacteria and Its Effect on Posthatch Salmonella Susceptibility. Poultry Science, 93, 818-829. http://dx.doi.org/10.3382/ps.2013-03409
[2] Manabe, K., Kageyama, Y., Morimoto, T., Shimizu, E., Takahashi, H., Kanaya, S., Ara, K., Ozaki, K. and Ogasawara, N. (2013) Improved Production of Secreted Heterologous Enzyme in Bacillus subtilis Strain MGB874 via Modification of Glutamate Metabolism and Growth Conditions. Microbial Cell Factories, 12, 18. http://dx.doi.org/10.1186/1475-2859-12-18
[3] Veenig, J.W., Smits, W.K., Hamoen, L.W. and Kuipers, O.P. (2006) Single Cell Analysis of Gene Expression Patterns of Competence Development and Initiation of Sporulation in Bacillus subtilis Grown on Chemically Defined Media. Journal of Applied Microbiology, 101, 531-541.
[4] Yu, X., Hallett, S.G., Sheppard, J. and Watson, A.K. (1998) Effects of Carbon Concentration and Carbon-to-Nitrogen Ratio on Growth, Conidiation, Spore Germination and Efficacy of the Potential Bioherbicide Colletotrichum coccodes. Journal of Industrial Microbiology and Biotechnology, 20, 333-338. http://dx.doi.org/10.1038/sj.jim.2900534
[5] Fisher, S.H. (1999) Regulation of Nitrogen Metabolism in Bacillus subtilis Vive la Différence! Molecular Microbiology, 32, 223-232. http://dx.doi.org/10.1046/j.1365-2958.1999.01333.x
[6] Carvalho, A.L.U., Oliveira, F.H.P.C., Mariano, R.L.R., Gouveia, E.R. and Souto-Maior, A.M. (2010) Growth, Sporulation and Production of Bioactive Compounds by Bacillus subtilis R14. Brazilian Archives of Biology and Technology, 53, 643-652.
[7] Dauner, M., Storni, T. and Sauer, U. (2001) Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture. Journal of Bacteriology, 183, 7308-7317. http://dx.doi.org/10.1128/JB.183.24.7308-7317.2001
[8] Slieman, T.A. and Nicholson, W.L. (2001) Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial an Solar UV Radiation. Applied and Environmental Microbiology, 67, 1274-1279. http://dx.doi.org/10.1128/AEM.67.3.1274-1279.2001
[9] O’Hara, M.B. and Hageman, J. (1990) Energy and Calcium Ion Dependence of Proteolysis during Sporulation of Bacillus subtilis Cells. Journal of Bacteriology, 172, 4161-4170.
[10] Hauser, P.M. and Errington, J. (1995) Characterization of Cell Cycle Events during the Onset of Sporulation in Bacillus subtilis. Journal of Bacteriology, 177, 3923-3932.
[11] Monteiro, S.M.S., Clemente, J.J., Henriques, A.O., Gomes, R.G., Carrondo, M.J.T. and Cunha, A.E. (2005) A Procedure for High-Yield Spore Production by Bacillus subtilis. Biotechnology Progress, 21, 1026-1031. http://dx.doi.org/10.1021/bp050062z
[12] Jolliffe, L.K., Doyle, R.J. and Streips, U.N. (1981) The Energized Membrane and Cellular Autolysis in Bacillus subtilis. Cell, 25, 753-763. http://dx.doi.org/10.1016/0092-8674(81)90183-5
[13] Hu, P., Leighton, T., Ishkhanova, G. and Kustu, S. (1999) Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria. Journal of Bacteriology, 181, 5042-5050.
[14] Barbosa, T.M., Serra, C.R., la Regione, R., Woodward, M.J. and Henriques, A.O. (2005) Screening for Bacillus Isolates in the Broiler Gastrointestinal Tract. Applied and Environmental Microbiology, 71, 968-978. http://dx.doi.org/10.1128/AEM.71.2.968-978.2005
[15] Jernejc, K., Cimerman, A. and Perdih, A. (1986) Comparison of Different Methods for Protein Determination in Aspergillus niger Mycelium. Applied Microbiology and Biotechnology, 23, 445-448. http://dx.doi.org/10.1007/BF02346058
[16] Reed, L.J. and Muench, H.A. (1938) Simple Method for Estimating Fifty Percent Endpoints. The American Journal of Hygiene, 27, 493-497.
[17] Ooijkass, L.P., Wilkinson, E.C., Tramper, J. and Buitelaar, R.M. (1998) Medium Optimization for Spore Production of Coniothyrium minitans Using Statistically—Based Experimental Designs. Biotechnology and Bioengineering, 64, 92-100.
[18] Tavares, M.B., Souza, R.D., Luiz, W.B., Cavalcante, R.C.M., Casaroli, C., Martins, E.G., Ferreira, R.C.C. and Ferreira, L.C.S. (2013) Bacillus subtilis Endospores at High Purity and Recovery Yields: Optimization of Growth Conditions and Purification Method. Current Microbiology, 66, 279-285. http://dx.doi.org/10.1007/s00284-012-0269-2

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.