Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from Bacillus thuringiensis subsp. kurstaki

DOI: 10.4236/abb.2014.510093   PDF   HTML   XML   3,421 Downloads   4,376 Views   Citations

Abstract

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.

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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.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[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.
http://dx.doi.org/10.1016/j.jbiosc.2013.05.008
[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.
http://dx.doi.org/10.4236/aim.2013.35062
[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. http://dx.doi.org/10.1126/science.1139208
[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.
http://dx.doi.org/10.4236/abb.2013.42032
[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.
http://dx.doi.org/10.1007/s00449-009-0391-0
[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.
http://dx.doi.org/10.4236/aer.2013.13005
[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. http://dx.doi.org/10.3109/10520299209110001
[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. http://dx.doi.org/10.1016/S0022-2011(02)00018-6
[11] Bartholomew, J.W. and Mittwer, T. (1950) A Simplified Bacterial Spore Stain. Biotechnic and Histochemistry, 25, 153-156.
http://dx.doi.org/10.3109/10520295009110979
[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. http://dx.doi.org/10.1111/j.1365-2672.2004.02184.x
[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.
http://dx.doi.org/10.1046/j.1472-765x.2002.01047.x
[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.
http://dx.doi.org/10.3923/jbs.2002.116.120
[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. http://dx.doi.org/10.1007/BF02716865
[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.
http://dx.doi.org/10.1016/S0032-9592(01)00198-4
[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. http://dx.doi.org/10.1002/jctb.1204
[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.
http://dx.doi.org/10.1007/s11270-007-9466-1
[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. http://dx.doi.org/10.1016/j.biortech.2010.12.098
[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. http://dx.doi.org/10.1590/S1516-89132011000100006
[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.
http://dx.doi.org/10.1007/s11274-007-9620-3
[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.
http://dx.doi.org/10.1021/pr4003728

  
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