Malays. Appl. Biol. (June 2003) 32(1): 61-67
MEDIUM FORMULATION FOR IMPROVEMENT OF GROWTH AND
SPORULATION OF BACILLUS THURINGIENSIS SUBSP. KURSTAKI
USING A TWO-STAGE CONTINUOUS CULTURE
WAN-MOHTAR, W.Y. and MOHD-MAZMIRA, M.M.
School of Bioscience and Biotechnology,
Faculty of Science and Technology,
Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia
ABSTRACT
Effect of glucose as carbon source, L-glutamate as amino acid source and hydrolysed vegetable protein (HVP) as nitrogen source on the production of Bacillus thuringiensis subsp. kurstaki SN5 were investigated in a two-stage continuous culture. Carbon, amino acid and nitrogen sources were found to increase growth in the first reactor and sporulation in the second reactor. The cultivation medium with 1% (w/v) glucose, 0.2 g/L L-glutamate and 0.5% (w/v) hydrolysed vegetable protein produced the highest total viable cell (TVCC) of 4.0 x 109 cells/ml in the first reactor and the highest spore count (SC) of 3.2 x 109 spores/ml in the second reactor. The highest spore percentage achieved (71%) was in the second reactor as compared to only 51% in the first reactor. The high spore percentage (71%) corresponds to a high 8-endotoxin value of 1706 mg/L. It was shown that under suitable conditions, high production of vegetative cells in the first reactor followed by high sporulation and sustained in the second reactor could be achieved.
ABSTRAK
Kesan kepekatan glukosa sebagai sumber karbon, L-glutamat sebagai sumber asid amino dan protein sayur terhidrolisis (HVP) sebagai sumber nitrogen terhadap penghasilan Bacillus thuringiensis subsp. kurstaki SN5 di dalam kultur selanjar dua tahap telah diselidiki. Kesan nutrien sumber karbon, asid amino dan sumber nitrogen didapati meningkatkan pembiakan sel di dalam reaktor pertama dan pensporaan di dalam reaktor kedua. Medium pertumbuhan yang mengandungi 1 % (w/ v) glukosa, 0.2 g/L L-glutamat dan 0.5% (w/v) HVP didapati memberikan nilai sel hidup viabel (TVCC) tertinggi dengan kepekatan 4.0 x 109 sel/ml di dalam reaktor pertama dan kiraan spora (SC) tertinggi di dalam reaktor kedua sebanyak 3.2 x 109 spora/mL. Nilai peratus spora tertinggi yang dicatat dalam reaktor kedua mencecah sehingga 71% berbanding hanya 51% dalam reaktor pertama. Peratus spora yang tinggi (71%) juga didapati berpadanan dengan kepekatan 8-endotoksin sebanyak 1706 mg/L. Dalam sekitaran tertentu, penghasilan sel-sel vegetatif didapati tinggi dalam reaktor pertama dan diikuti dengan pensporaan tinggi dan mampan dalam reaktor kedua dapat diselenggarakan.
Key words: Two-stage continuous culture, medium formulation, 5-endotoxin.
REFERENCES
Adams, T.T., Eiteman, M.A. and Adang, M.J. 1999. Bacillus thuringiensis subsp. kurstaki spore production in batch culture using broiler litter extracts as complex media. Bioresource Technology, 67: 83-87.
Bechtel, D.B. and Bulla, L.A. 1976. Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. Bacteriology 127 (3): 1472-1481.
Beegle, C.C., Rose, R.I. and Ziniu, Y. 1987. Mass production of Bacillus thuringiensis and B. sphaericus for microbial control of insect pests.
In: Biotechnology for Biological Control of Pests and Vectors, Maramorosch, K. (Eds.),
Florida: CRC Press, pp. 195-216. Bulla, L.A. and Rhodes, A. 1975. Bacteria as insect
pathogens. Annual Review of Microbiology, pp. 37-47.
Doi, R.H. 1989. Sporulation and germination. In: Biotechnology handbooks 2: Bacillus, Colin R.H. (Eds.). New York: Plenum Press, pp. 169-170.
Ejiofor, A.O. and Okafor, N. 1989. Production of mosquito larvicidal Bacillus thuringiensis serotype H-14 on raw material media from Nigeria. Journal of Applied. Bacteriology, 67: 5-9.
Egorov, N.S., Loriya, Zh.K. and Yudina, T.G. 1981. Relationship between spore formation and exoproteinase synthesis. Microbiology, 49 (4): 534-539.
Hannay, C.L. 1953. Crystalline inclusion in aerobic spore-forming bacteria. Nature (London) 172: 1004
Kang, B.C., Lee, S.Y. and Chang, H.N. 1992. Enhanced spore production of Bacillus thuringiensis by fed batch culture. Biotechnology Letters, 14 (8): 721-726.
Kang, B.C., Lee, S.Y. and Chang, H.N. 1993. Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system. Biotechnology Bioengineering, 42: 1107-1112.
Khovrychev, M.P., Sakharova, Z.V., Ignatenko, Y.N., Blokhina, T.P. and Robotnova, I.L. 1987. Spore formation and biosynthesis of protein crystals during continuous culturing of Bacillus thuringiensis. Microbiology, 55 (6): 790-795. Translated from Mikrobiologiya, 55 (6): 983-988.
Khovrychev, M.P., Slobodkin, A.N., Sakharova, Z.V. and Blokhina, T.P. 1991. Growth and development of Bacillus thuringiensis in multiple stage continuous culture. Microbiology, 59 (6): 695-698. Translated from Mikrobiologiya, 59 (6): 998-1003.
Kuznetsov, L.E., Khovrychev, M.P. and Sakharova, Z.V. 1984. A synthetic medium for
