Abstract
To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90°C for 1 h, showed that spores produced under a sublethal temperature of 57°C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37°C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15–25°C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 μm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beaman TC, Gerhardt P (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl Environ Microbiol 52:1242–1246
Bernhardt J, Völker U, Völker A, Antelmann H, Schmid R, Mach H, Hecker M (1997) Specific and general stress proteins in Bacillus subtilis—a two dimentional electrophoresis study. Microbiology 143:999–1017
Brachman PS, Freely JC (1970) Spore stain (Wirts–Conklin). In: Blair JE, Lennette EH, Truant JP (eds) Manual of clinical microbiology. ASM, Bethesda, MD
Burges HD, Jones KA (1998) Trends in formulation of microorganisms and future research requirements. In: Burges HD (ed) Formulation of microbial biopesticides—beneficial microorganisms, nematodes and seed treatments. Kluwer, London, pp 311–332
Carrera M, Zandomeni RO, Fitsgibbon J, Sagripanti J-L (2007) Difference between the spore sizes of Bacillus antracis and other Bacillus species. J Appl Micrbiol 102:303–312
Chung S, Kim S-D (2005) Biological control of phytopathogenic fungi by Bacillus amyloliquefaciens 7079; suppression rates are better than popular chemical fungicides. J Microbiol Biotechnol 15:1011–1021
Dandurand LM, Morra MJ, Chaverra MH, Orser CS (1994) Survival of Pseudomonas spp. in air-dried mineral powders. Soil Biol Biochem 26:1423–1430
Fairhead H, Setlow B, Setlow P (1993) Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species. J Bacteriol 175:1367–1374
Hecker MW, Schumann W, Völker U (1996) Heat shock and general stress response in Bacillus subtilis. Mol Microbiol 19:417–428
Heredia NL, Garcia GA, Luevanos R, Labbe RG, Sontos JS (1997) Evaluation of the heat resistance of vegetative cells and spores of Clostridium perfringens type A by sublethal heat shock. J Food Prot 60:998–1000
Jackson MA, Cliquet S, Iten LB (2003) Media and fermentation processes for the rapid production of high concentrations of stable blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus. Biocontrol Sci Technol 13:23–33
Jeong H-K, Kim J-R, Woo S-M, Kim S-D (2006) An auxin producing plant growth promoting rhizobacterium Bacillus subtilis AH18 which has siderophore-producing biocontrol activity. Kor J Microbiol Biotechnol 94:94–100
Jones KA, Burges HD (1998) Technology of formulation and application. In: Burges HD (ed) Formulation of microbial biopesticides—beneficial microorganisms, nematodes and seed treatments. Kluwer, London, pp 7–30
Kim S-H, Kwon J-S (2001) Methods to improve soil microbial fertilizers. In: Soil microbial fertilizers. Korea Rural Development Administration, Republic of Korea, pp 47–63
Lee J-H (2001) Present state of commercialization of soil microbial fertilizers in South Korea. In: Soil microbial fertilizers. Korea Rural Development Administration, Republic of Korea, pp 31–45
Lim HM, Lee SH, Park J-B, Kwon J-A, Yu Y-S (2006) Correlation of physical properties of inorganic carrier and microbial survival rate. Key Eng Mater 317–318:793–796
Lowell S, Shields JE (1984) Powder surface area and porosity. In: Scarlett B (ed) Powder technology series, 3rd edn. Chapman & Hall, London, pp 119–120
Movahedi S, Waites W (2000) A two dimensional protein gel electorphoresis study of the heat stress response of Bacillus subtilis cells during sporulation. J Bacteriol 182:4758–4763
Periago PA, van Schaik W, Abee T, Wouters JA (2002) Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14679. Appl Environ Microbiol 68:3486–3495
Ruthvan DM (1984) Principles of adsorption and adsorption processes. Wiley, New York, pp 1–61
Sedlák MV, Vinter V, Adamec J, Vohradsky J, Voburka Z, Chaloupka J (1993) Heat shock applied early in sporulation affects heat resistance Bacillus megaterium spores. J Bacteriol 175:8049–8052
Slininger PJ, Van Cauwenberge JE, Bothast RJ, Weller DM, Thomashow LS, Cook RJ (1996) Effect of growth culture physiological state, metabolites, and formulation on the viability, phytotoxicity, and efficacy of the take-all biocontrol agent Pseudomonas fluorescens 2–79 stored encapsulated on wheat seeds. Appl Microbiol Biotechnol 45:391–398
Acknowledgments
Authors are grateful to Jun-Hyung Hong for laboratory assistance and to Kyung-Min Kim for generating the survival curves. This research was funded by the Korean Rural Development Administration, Biogreen 21 project, grant number 105649.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chung, S., Lim, H.M. & Kim, SD. Formulation of stable Bacillus subtilis AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers. Appl Microbiol Biotechnol 76, 217–224 (2007). https://doi.org/10.1007/s00253-007-0992-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-0992-y