Applied Microbiology and Biotechnology

, Volume 86, Issue 2, pp 499–508 | Cite as

A downstream process for production of a viable and stable Bacillus cereus aquaculture biological agent

  • Rajesh Lalloo
  • Dheepak Maharajh
  • Johann Görgens
  • Neil Gardiner
Biotechnological Products and Process Engineering


Biological products offer advantages over chemotherapeutics in aquaculture. Adoption in commercial application is lacking due to limitations in process and product development that address key end user product requirements such as cost, efficacy, shelf life and convenience. In previous studies, we have reported on the efficacy, physiological robustness and low-cost spore production of a Bacillus cereus isolate (NRRL 100132). This study examines the development of suitable spore recovery, drying, formulation and tablet production from the fermentation product. Key criteria used for such downstream process unit evaluation included spore viability, recovery, spore balance, spore re-germination, product intermediate stability, end product stability and efficacy. A process flow sheet comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded a suitable product. The formulation included corn steep liquor and glucose to enhance subsequent spore re-germination. Viable spore recovery and spore balance closure across each of the process units was high (>70% and >99% respectively), with improvement in recovery possible by adoption of continuous processing at large scale. Spore re-germination was 97%, whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves. The process resulted in a commercially attractive product and suitable variable cost of production.


Bacillus cereus Downstream processing Biological agent Aquaculture 



We thank Reshnee Baboolall, Swasthi Soomaroo and Arvesh Parsoo for technical assistance and BioPAD Biotechnology Regional Innovation Centre for funding.


  1. Amer GA, Utkhede RS (2000) Development of formulations of biological agent for management of root rot of lettuce and cucumber. Can J Microbiol 43:809–816CrossRefGoogle Scholar
  2. Aoyama Y, Shigeta Y, Okazaki T, Hagura Y, Suzuki K (2005) Non-thermal inactivation of Bacillus spores by pressure-holding. Food Sci Technol Res 11:324–327CrossRefGoogle Scholar
  3. Bayrock D, Ingledew WM (1997) Mechanism of viability loss during fluidized bed drying of baker’s yeast. Food Res Int 30:417–425CrossRefGoogle Scholar
  4. Biourge V, Vallet C, Levesque A, Sergheraert R, Chevalier S, Robertson JL (1998) The use of probiotics in the diet of dogs. J Nutri 128:2730S–2732SGoogle Scholar
  5. Brar SK, Verma M, Tyagi RD, Valéro JR (2006) Recent advances in downstream processing and formulations of Bacillus thuringiensis based on biopesticides. Process Biochem 41:323–342CrossRefGoogle Scholar
  6. Busscher HJ, Weerkamp AH (1987) Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Microbiol Rev 46:165–173CrossRefGoogle Scholar
  7. Chen XD, Patel KC (2007) Microorganism inactivation during drying of small droplets or thin-layer slabs—a critical review of existing kinetics models and an appraisal of the drying rate dependent model. J Food Eng 82:1–10CrossRefGoogle Scholar
  8. Costa E, Teixidό N, Usall J, Fons E, Gimeno V, Delgado J, Viñas I (2001) Survival of Pantoea agglomerans strain CPA-2 in spray-drying process. J Food Protection 65:185–191Google Scholar
  9. Dandurand LM, Morra MJ, Chaverra MH, Orser CS (1994) Survival of Pseudomonas spp. in air-dried mineral powders. Soil Biol Biochem 26:1423–1430CrossRefGoogle Scholar
  10. de Medeiros FPM, de Melo Santos MAV, Regis L, Rios EMM, Neto PJM (2005) Development of a Bacillus sphaericus tablet formulation and its evaluation as a larvicide in the biological control of Culex quinquefasciatus. Mem Inst Oswaldo Cruz 100:431–434CrossRefGoogle Scholar
  11. Driks A (2004) The Bacillus spore coat. Phytopathology 94:1249–1251CrossRefGoogle Scholar
  12. Emmert EAB, Handelsman J (1999) Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol Lett 171:1–9CrossRefGoogle Scholar
  13. Fast AW, Menasveta P (2000) Some recent issues and innovations in marine shrimp pond culture. Rev Fish Sci 8:151–233CrossRefGoogle Scholar
  14. Gatesoupe FJ (1999) The use of probiotics in aquaculture. Aquaculture 180:147–165CrossRefGoogle Scholar
  15. Guetsky R, Shtienberg Y, Elad Y, Fischer E, Dinoor A (2002) Improving biological control by combining biocontrol agents each with several mechanisms of disease suppression. Phytopathology 92:976–985CrossRefGoogle Scholar
  16. Hong HA, Duc LH, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 29:813–835CrossRefGoogle Scholar
  17. Keller K, Friedmann T, Boxman A (2001) The bioseparation needs for tomorrow. Trends Biotechnol 19:438–441CrossRefGoogle Scholar
  18. Klein N, Lortal S (1999) Attenuated starters: an efficient means to influence cheese ripening—a review. Int Dairy J 9:751–762CrossRefGoogle Scholar
  19. Lalloo R, Ramchuran S, Ramduth D, Görgens J, Gardiner N (2007) Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. J Appl Microbiol 103:1471–1479CrossRefGoogle Scholar
  20. Lalloo R, Maharajh D, Görgens J, Gardiner N (2008) Functionality of a Bacillus cereus biological agent in response to physiological variables encountered in aquaculture. Appl Microbiol Biotechnol 79:111–118CrossRefGoogle Scholar
  21. Lalloo R, Maharajh D, Görgens J, Gardiner N (2009) High-density spore production of a B. cereus aquaculture biological agent by nutrient supplementation. Appl Microbiol Biotechnol 83:59–66CrossRefGoogle Scholar
  22. Larena I, de Cal A, Liñán M, Melgarejo P (2003) Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease. J Appl Microbiol 94:508–514CrossRefGoogle Scholar
  23. Luna-Solano G, Salgado-Cervantes MA, Rodríguez-Jimenes GC, García-Alvarado MA (2005) Optimization of brewer’s yeast spray drying process. J Food Eng 6:89–18Google Scholar
  24. Margosch D, Gänzle MG, Erhmann MA, Vogel RF (2004) Pressure inactivation of Bacillus endospores. Appl Environ Microbiol 70:7321–7328CrossRefGoogle Scholar
  25. Mathys A, Heinz V, Knorr D (2008) New pressure and temperature effects on bacterial spores. J Phys Conf Ser 121:1–5CrossRefGoogle Scholar
  26. Mille Y, Obert J, Beney L, Gervais P (2004) New drying process for lactic bacteria based on their dehydration behaviour in liquid medium. Biotechnol Bioeng 88:71–76CrossRefGoogle Scholar
  27. Moënne-Loccoz Y, Naughton M, Higgins P, Powell J, O’Connor B, O’Gara F (1999) Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113. J Appl Microbiol 86:108–116CrossRefGoogle Scholar
  28. Moriarity DJW (1999) Disease control in shrimp aquaculture with probiotic bacteria. Microbial interactions in aquaculture. In: Bell CR, Brylinsky M (eds) Proceedings of the 8th International Symposium on Microbial Ecology, CanadaGoogle Scholar
  29. Prabakaran G, Hoti SL (2008) Application of different downstream processing methods and their comparison for the large-scale preparation of Bacillus thuringiensis var. israelensis after fermentation for mosquito control. Biologicals 36:412–415CrossRefGoogle Scholar
  30. Puziss M, Manning LC, Lynch JW, Barclay E, Abelow I, Wright GG (1963) Large-scale production of protective antigen of Bacillus anthracis in anaerobic cultures. Appl Microbiol 11:330–334Google Scholar
  31. Ramsamy HS, Singh RP (1997) Sterilization process engineering. In: Valentas KJ, Rotstein E, Singh RP (eds) Handbook of food engineering practice. CRC Press, New York, pp 39–42Google Scholar
  32. Rivière J (1977) Industrial applications of microbiology. Wiley, LondonGoogle Scholar
  33. Rojas JV, Gutierrez E, De la Torre M (1996) Primary separation of the entomopathogenic products of Bacillus thuringiensis. Biotechnol Prog 12:564–566CrossRefGoogle Scholar
  34. Rönner U, Husmark U, Henriksson A (1990) Adhesion of Bacillus spores in relation to hydrophobicity. J Appl Bacteriol 69:550–556Google Scholar
  35. Rowe GE, Margaritis A (2004) Bioprocess design and economic analysis for the commercial production of environmentally friendly bio-insecticides from Bacillus thuringiensis HD-1 kurstaki. Biotechnol Bioeng 86:377–388CrossRefGoogle Scholar
  36. Sanders ME, Morelli L, Tompkins TA (2003) Spore formers as human probiotics: Bacillus, Sporolactobacillus and Brevibacillus. Comp Rev Food Sci Safety 2:101–110CrossRefGoogle Scholar
  37. Schisler DA, Slininger PJ, Behle RW, Jackson MA (2004) Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology 94:1267–1271CrossRefGoogle Scholar
  38. Setlow P (2006) Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101:514–525CrossRefGoogle Scholar
  39. Setlow B, Setlow P (1995) Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat. Appl Environ Microbiol 61:2787–2790Google Scholar
  40. Soper RS, Ward MG (1981) Beltsville symposia in agricultural research. Biol Cont Crop Production 5:161–180Google Scholar
  41. Tamez-Guerra P, McGuire MR, Medrano-Roldan H, Galan-Wong LJ (1996) Sprayable granule formulations of Bacillus thuringiensis. Biotechnol Prog 12:564–566CrossRefGoogle Scholar
  42. Torres-Anjel MJ, Hedrick TI (1970) Spore removal by centrifugation and its effect on ultra-high temperature commercial sterilization of milk. J Dairy Res 54:326–330Google Scholar
  43. Tsun HY, Liu CM, Tzeng YM (1999) Recovery and purification of thuringiensin from the fermentation broth of Bacillus thuringiensis. Bioseparation 7:309–316CrossRefGoogle Scholar
  44. Van Dam-Mieras MCE, de Jeu WH, de Vries J, Curell BR, James JW, Leach CK, Patmore RA (1995) Product recovery in bioprocess technology (Biotol). Butterworth-Heinemann, OxfordGoogle Scholar
  45. Werner L, Latzko F, Hampel W (1993) Spray drying of yeast-lytic enzymes from Arthrobacter sp. Biotechnol Tech 7:663–666CrossRefGoogle Scholar
  46. Wiwattanapatapee R, Pengnoo A, Kanjanamaneesathian M, Matchavanich W, Nilratana L, Jantharangsri A (2004) Floating pellets containing bacterial antagonists for control sheath blight of rice: formulations, viability and bacterial release studies. J Control Release 95:455–462CrossRefGoogle Scholar
  47. Zamola B, Valles P, Meli G, Miccoli P, Kajfez F (1981) Use of the centrifugal separation technique in manufacturing a bioinsecticide based on Bacillus thuringiensis. Biotechnol Bioeng 23:1079–1086CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Rajesh Lalloo
    • 1
    • 2
  • Dheepak Maharajh
    • 1
    • 2
  • Johann Görgens
    • 2
  • Neil Gardiner
    • 1
  1. 1.CSIR BiosciencesModderfonteinSouth Africa
  2. 2.Department of Process EngineeringStellenbosch UniversityStellenboschSouth Africa

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