Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Bioindicator production with Bacillus atrophaeus’ thermal-resistant spores cultivated by solid-state fermentation


Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Association for the Advancement of Medical Instrumentation (AAMI) (2005) ST40:2004. Dry heat (heated air) sterilizers. American National Standard, Arlington

  2. Capalbo DM, Valicente FH, Moraes IO, Pelizer LH (2001) Solid-state fermentation of Bacillus thuringiensis tolworthi to control fall armyworm in maize. Electron J Biotechnol 4:9–10

  3. Cazemier AE, Wagenaars SFM, Steeg PF (2001) Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli. J Appl Microbiol 90:761–777

  4. Chen X, Chen S, Su M, Yu Z (2005) High yield of poly-γ-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate. Bioresour Technol 96:1872–1879

  5. Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1:117–126

  6. Goes AP, Sheppard JD (1999) Effect of surfactants on α-amylase production in a solid substrate fermentation process. J Chem Technol Biotechnol 74:709–712

  7. Gupta S, Kapoor M, Sharma K, Nair L, Kuhad RC (2008) Production and recovery of an alkaline exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation using statistical approach. Bioresour Technol 99:937–945

  8. Kashyap DR, Soni SK, Tewari R (2003) Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation. Bioresour Technol 88:251–254

  9. Kunamneni A, Singh S (2005) Response surface optimization of enzymatic hydrolysis of maize starch for higher glucose production. Biochem Engin J 27:179–190

  10. Montgomery DC (1977) Response surface methods and other approaches to process optimization. In: Montgomery DC (ed) Design and analysis of experiments. Wiley, New York, pp 427–510

  11. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr Sci 77:149–162

  12. Piggot PJ, Losick R (2002) Sporulation genes and intercompartmental regulation. In: Sonenshein AL, Losick R, Hoch JA (eds) Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, DC, pp 483–517

  13. Pinto TJ, Saito T (1992) Esterilização por óxido de etileno. II—Influência de corpos de prova no desempenho de monitores biológicos e sua avaliação. Rev Saúde Pública 26:379–383

  14. Pryor SW, Gibson DM, Hay AG, Gossett JM, Walker LP (2007) Optimization of spore and antifungal lipopeptide production during the solid state fermentation of Bacillus subtilis. Appl Biochem Biotechnol 143:63–79

  15. Redmond C, Baillie LW, Hibbs S, Moir AJ (2004) Identification of proteins in the exosporium of Bacillus anthracis. Microbiology 150:355–363

  16. Schaeffer PJ, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci USA 54:704–711

  17. Sella SRBR, Vandenberghe LPS, Medeiros ABP, Soccol CR (2007) Evaluation of different techniques and culture medium for the Bacillus atrophaeus spores production. Proceedings of the Anais XVI Simpósio Nacional de Bioprocessos, Curitiba, Brazil, CD ISSN/ISBN:9788560328079

  18. Soares VF, Castilho LR, Bon EPS, Freire DMG (2005) High-yield Bacillus subtilis protease production by solid-state fermentation. Appl Biochem Biotechnol 121:311–320

  19. Soccol CR, Vandenberghe LPS (2003) Overview of applied solid-state fermentation in Brazil. Biochem Eng J 13:205–218

  20. Soccol CR, Radjiskumar M, Quorim M (2001) Uso do bagaço de cana de açúcar como material de suporte na fase de enraizamento em processo de micropropagação vegetal. Brazilian patent, NPI No. 931

  21. Sonenshein AL (2000) Control of sporulation initiation in Bacillus subtilis. Curr Opin Microbiol 3:561–566

  22. United States Pharmacopeia XXIX (2005) Biological indicators resistance and performance tests. In: The United States Pharmacopeia, 29th rev. United States Pharmacopoeia Convection Rockville, MD, pp 2501–2503

  23. Veening J (2007) Phenotypic variation in Bacillus subtilis: bistability in the sporulation pathway. Proefschrift Rijksuniversiteit Groningen, Nederland, Print Partners Ipskamp, Enschede

  24. Vries YP, Atmadja RD, Hornstra LM, deVos WM, Abee T (2005) Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in a defined medium. Appl Environ Microbiol 71:3248–3254

Download references


This research was financially supported by the Secretaria de Estado da Ciência, Tecnologia e Ensino Superior—Fundo Paraná.

Author information

Correspondence to Carlos Ricardo Soccol.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sella, S.R.B.R., Guizelini, B.P., Vandenberghe, L.P.S. et al. Bioindicator production with Bacillus atrophaeus’ thermal-resistant spores cultivated by solid-state fermentation. Appl Microbiol Biotechnol 82, 1019–1026 (2009). https://doi.org/10.1007/s00253-008-1768-8

Download citation


  • Bacillus atrophaeus
  • Bioindicator
  • Solid-state fermentation
  • Sugar cane bagasse
  • Response surface methodology
  • Optimization