ABB  Vol.5 No.10 , September 2014
Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from Bacillus thuringiensis subsp. kurstaki
Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt); and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h; while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores; malachite green-saffranin stained both δ-crystals and endospores; and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

Cite this paper
Jisha, V. and Benjamin, S. (2014) Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from Bacillus thuringiensis subsp. kurstaki. Advances in Bioscience and Biotechnology, 5, 797-804. doi: 10.4236/abb.2014.510093.
[1]   Smitha, R.B., Jisha, V.N., Pradeep, S., Sarath Josh, M.K. and Benjamin, S. (2013) Potato Flour Mediated Solid-State Fermentation for the Enhanced Production of Bt-Toxin. Journal of Bioscience and Bioengineering, 116, 595-601.

[2]   Jisha, V.N., Smitha, R.B. and Benjamin, S. (2013) An Overview on the Crystal Toxins from Bacillus thuringiensis. Advances in Microbiology, 3, 462-472.

[3]   Marvier, M., Creedy, C.M., Regetz, J. and Karieva, P. (2007) A Meta-Analysis of Effects of Bt Cotton and Maize on Nontarget Invertebrates. Science, 316, 1475-1477.

[4]   Benjamin, S., Smitha, R.B., Jisha, V.N., Pradeep, S., Sajith, S., Sreedevi, S., Priji, P., Unni, K.N. and Sarath Josh, M.K. (2013) A Monograph on Amylases from Bacillus spp. Advances in Bioscience and Biotechnology, 4, 227-241.

[5]   Vu, K.D., Tyagi, R.D., Valéro, J.R. and Surampalli, R.Y. (2010) Batch and Fed-Batch Fermentation of Bacillus thuringiensis Using Starch Industry Wastewater as Fermentation Substrate. Bioprocess and Biosystems Engineering, 33, 691-700.

[6]   Benjamin, S. and Pandey, A. (1998) Candida rugosa Lipases: Molecular Biology and Versatility in Biotechnology. Yeast, 14, 1069-1087.

[7]   Jisha, V.N., Smitha, R.B., Pradeep, S., Sreedevi, S., Unni, K.N., Sajith, S., Priji, P., Sarath Josh, M.K. and Benjamin, S. (2013) Versatility of Microbial Proteases. Advances in Enzyme Research, 1, 39-51.

[8]   Schichnes, D., Nemson, J.A. and Ruzin, S.E. (2006) Fluorescent Staining Method for Bacterial Endospores. Microscope, 54, 91-93.

[9]   Sharma, D.K. and Prasad, D.N. (1992) Rapid Identification of Viable Bacterial Spores Using a Fluorescence Method. Biotechnic and Histochemistry, 67, 27-29.

[10]   Rampersad, J., Khan, A. and Ammons, D. (2002) Usefulness of Staining Parasporal Bodies When Screening for Bacillus thuringiensis. Journal of Invertebrate Pathology, 79, 203-204.

[11]   Bartholomew, J.W. and Mittwer, T. (1950) A Simplified Bacterial Spore Stain. Biotechnic and Histochemistry, 25, 153-156.

[12]   Laflamme, C., Lavigne, S., Ho, J. and Duchaine, C. (2004) Assessment of Bacterial Endospore Viability with Fluorescent Dyes. Journal of Applied Microbiology, 96, 684-692.

[13]   Tortora, G.J., Funke, B.R. and Christine, L. (2004) Microbiology, an Introduction. 8th Edition. Pearson Education Inc., Upper Saddle River and Dorling Kindersley Publishing Inc., London.

[14]   Setlow, P. (2000) Resistance of Bacterial Spores. In: Storz, G. and Hengge-Aronis, R., Eds., Bacterial Stress Responses, American Society for Microbiology, Washington DC, 217-230.

[15]   Liu, W.M. and Bajpai, R.K. (1995) A Modified Growth Medium for Bacillus thuringiensis. Biotechnolology, 11, 589-591.

[16]   Hamouda, T., Shih, A.Y. and Baker, J.R. (2002) A Rapid Staining Technique for the Detection of the Initiation of Germination of Bacterial Spores. Letters in Applied Microbiology, 34, 86-90.

[17]   Chilcott, C.N., Wigley, P.J., Broadwell, A.H., Park, D.J. and Ellar, D.J. (1998) Activities of Bacillus thuringiensis Insecticidal Crystal Proteins Cyt1Aa and Cyt2Aa against Three Species of Sheep Blowfly. Applied Environmental Microbiology, 64, 4060-4061.

[18]   Fadel, M. and Sabour, M. (2002) Utilisation of Diary Byproduct in the Production of Bioinsecticide. Journal of Biological Science, 2, 116-120.

[19]   Prescott, L., Harley, J. and Klein, D. (1996) Microbiology. 3rd Edition, Wm.C. Brown Publishers, Dubuque.

[20]   Chestukhina, G.G., Zalunin, I.A., Kostina, L.I., Kotova, T.S., Kattrukha, S.P. and Stepanov, V. (1980) Crystal Forming Proteins of Bacillus thuringiensis. Journal of Biochemisty, 187, 457-465.

[21]   Rajalakshmi, S. and Shethna, Y.I. (1980) Spore and Crystal Formation in Bacillus thuringiensis var. thuringiensis during Growth in Cystine and Cysteine. Journal of Bioscience, 2, 321-328.

[22]   Teixeira, M.B. (2012) Using Sewage Sludge from Municipal and Industrial Solid Wastes to Produce a Bacillus thuringiensis Biopesticide.

[23]   Lachhab, K., Tyagi, R.D. and Valéro, J.R. (2001) Production of Bacillus thuringiensis Biopesticides Using Wastewater Sludge as a Raw Material: Effect of Inoculum and Sludge Solids. Process Biochemistry, 37, 197-208.

[24]   Yezza, A., Tyagi, R.D., Valéro, J.R. and Surampalli, R.Y. (2005) Production of Bacillus thuringiensis Based Biopesticides by Batch and Fed-Batch Culture Using Wastewater Sludge as a Raw Material. Journal of Chemical Technology and Biotechnology, 80, 502-510.

[25]   Chang, M., Zhou, S., Lu, N. and Ni, J. (2007) Enhanced Bacillus thuringiensis Production from Sewage Sludge with Alkaline and Ultrasonic Pretreatments. Water, Air, and Soil Pollution, 186, 75-84.

[26]   Zhuang, L., Zhou, S., Wang, Y., Liu, Z. and Xu, R. (2011) Cost-Effective Production of Bacillus thuringiensis Biopesticides by Solid-State Fermentation Using Wastewater Sludge: Effects of Heavy Metals. Bioresource Technology, 102, 4820-4826.

[27]   da Silva, M., Furijo Jr., A, Furlan, A.S. and Souza, O. (2011) Production of Bio-Inseticide Bacillus thuringiensis var. israelensis in Semi Continuous Processes Combined with Batch Processes for Sporulation. Brazilian Archives of Biology and Technology, 54, 45-52.

[28]   Sarrafzadeh, M.H. and Navarro, J.M. (2006) The Effect of Oxygen on the Zsporulation, Delta-Endotoxin Synthesis and Toxicity of Bacillus thuringiensis H-14. World Journal of Microbiology and Biotechnology, 22, 305-310.

[29]   Das, J. and Danker, T. (2008) Microbial Population Dynamics, Especially Stress Tolerant Bacillus thuringiensis, in Partially Anaerobic Rice Field Soils during Post-Harvest Period of the Himalayan, Island, Brackish Water and Coastal Habitats of India. World Journal of Microbiology and Biotechnology, 24, 1403-1410.

[30]   Wang, J., Mei, H., Qian, H., Tang, Q., Liu, X., Yu, Z. and He, J. (2013) Expression Profile and Regulation of Spore and Parasporal Crystal Formation-Associated Genes in Bacillus thuringiensis. Journal of Proteome Research, 12, 5487-5501.