Development of a Cost Effective Bioprocess for Production of an Iranian Anti-Coleoptera Bacillus thuringiensis Strain

Authors
G. Salehi Jouzani
S. Abbasalizadeh
M. Mohammad Fata
H. Morsali
Department of Microbial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Islamic Republic of Iran.
Abstract
One of the major obstacles of Bt production as a biopesticide is its expensive bioprocess and fermentation. Therefore, the objective of the present study was to optimize growth condition and develop a low-cost bioprocess for mass production of a native coleopteran-effective Bacillus thuringiensis (Bt) strain (KH4) based on agricultural wastes, at incubator and batch fermenter level. Preliminary experiments showed that the optimum pH and temperature for the strain were 6.5 and 30°C, respectively. The maximum growth and spore/crystal production were observed in the medium containing 2% molasses and 3% corn steep liquor as carbon and nitrogen sources, respectively. Different concentrations of the sea salt were used as a new cheap and available mineral source. Sea salt with final 0.003% w/w concentration showed the highest rate of growth for the strain. The experiments in Batch fermenter showed that volume of 2% bacterial inoculation in total volume of medium culture was the best concentration as preculture. It was shown that pH significantly decreased at the beginning of logarithmic phase, whereas it significantly increased at the end of the logarithmic phase. By increasing fermentation period, the oxygen demand was increased, and by increasing oxygen concentration up to 70%, the bacterial growth and the spores/crystal production was increased. Based on the results, the growth condition of the strain was optimized and a new cheap and available commercial fermentation medium was developed for mass production of the strain in the batch systems.

Keywords

1. Adjalle, K. D., Brar, S. K., Verma, M., Tyagi, R. D., Valero, J. R. and Surampalli, R. Y. 2007. Ultrafiltration Recovery of Entomotoxicity from Supernatant of Bacillus thuringiensis Fermented Wastewater and Wastewater Sludge. Process. Biochem., 42: 1302-1311.
2. Amin, G., Alotaibi, S., Narmen, A. Y. and Saleh, W. D. 2008. Optimization of a Fermentation Process for Bioinsecticide Production by Bacillus thuringiensis. World J. Microbiol. Biotechnol., 24: 2465-2471.
3. Anderson, R. K. I. and Jayaraman, K. 2003. Influence of Carbon and Nitrogen Sources on the Growth and Sporulation of Bacillus thuringiensis var. galleriae for Biopesticide Production. Chem. Biochem. Eng., 17: 225-232.
4. Avignone-Rossa, C., Arcas, J. and Mignone, C. 1992. Bacillus thuringiensis Growth, Sporulation and δ-Endotoxin Production in Oxygen Limited and Non-limited Cultures. World J. Microbiol. Biotechnol., 8: 301-304.
5. Avignone-Rossa, C. and Mignone C. F. 1995. Bacillus thuringiensis Growth and Toxicity. Mol. Biotechnol., 4: 55-71.
6. Berbert-Molina, M. A., Prata, A. M. R., Pessanha, L. G. and Silveira, M. M. 2008a. Kinetics of Bacillus thuringiensis var. israelensis Growth on High Glucose Concentrations. J. Ind. Microbiol. Biotechnol. 35:1397–1404
7. Berbert-Molina, M. A., Prata, A. M. R., Pessanha, L. G. and Silveira, M. M. 2008b. Kinetics of Bacillus thuringiensis var. israelensis Growth on High Glucose Concentrations. J. Ind. Microbiol. Biotechnol., 35: 1397-1404.
8. Brar, S. K., Verma, M., Barnabéa, S. and Tyagi, R. D. 2005a. Impact of Tween 80 during Bacillus thuringiensis Fermentation of Wastewater Sludges. Process. Biochem., 40: 2695-2705.
9. Brar, S. K., Verma, M., Tyagi, R. D., Valéro, J. R. and Surampalli, R. Y. 2005b. Starch Industry Wastewater-based Stable Bacillus thuringiensis Liquid Formulations. J. Econom. Entomol., 98: 1890-1898.
10. Brar, S. K., Verma, M., Tyagi, R. D., Valéro, J. R. and Surampalli, R.Y. 2005c. Sludge Based Bacillus thuringiensis Biopesticides: Viscosity Impacts. Water Res., 39: 3001-3011.
11. Brownbridge, M. and Margalit, J. 1986. New Bacillus thuringiensis Strains Isolated in Israel Are Highly Toxic to Mosquito Larvae. J. Invertebr. Pathol. 48: 216–222.
12. Chang, M., Zhou, S. G., Lu, N. and Ni, J. R. 2008. Starch Processing Wastewater as a New Medium for Production of Bacillus thuringiensis. World J. Microbiol. Biotechnol., 24: 441–447.
13. Ghribi, D., Zouari, N., Trabelsi, H. and Jaoua, S. 2007a. Improvement of Bacillus thuringiensis δ-Endotoxin Production by Overcome of Carbon Catabolite Repression through Adequate Control of Aeration. Enzyme Microbial. Technol., 40: 614–622.
14. Ghribi, D., Zouari, N., Trigui, W. and Jaoua, S. 2007b. Use of Sea Water as Salts Source in Starch and Soya Bean-based Media, for the Production of Bacillus thuringiensis Bioinsecticides. Process. Biochem., 42: 374-378.
15. Huang, K., Badger, M., Haney, K. and Evans, S. L. 2007. Large Scale Production of Bacillus thuringiensis PS149B1 Insecticidal Proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens. Protein Expr. Purif., 53: 325-330.
16. Keshavarzi, M., Salimi, H., Mirzanamadi, F. 2005. Biochemical and Physical Requirements of Bacillus thuringiensis subsp. Kurstaki for High Biomass Yield Production. J. Agric. Sci. Technol. 7: 41-47.
17. Khojan, S., Keshavarzi, M., Zargari, K., Abdolahi, H. and Rouzbeh, F. 2013. Presence of Multiple Cry Genes in Bacillus thuringiensis Isolated from Dead Cotton Bollworm Heliothis armigera. Production. J. Agric. Sci. Technol., 15: 1285-1295.
18. Khodair, T. A., Abdelhafez, A. A. M., Sakr, H. M. and Ibrahim M. M. M. 2008. Improvement of Bacillus thuringiensis Bioinsecticide Production by Fed-batch Culture on Low Cost Effective Medium. Res. J. Agri. Biol. Sci., 4: 923-935.
19. Kraemer-Schafhalter, A. and Moser, A. 1996. Kinetic Study of Bacillus thuringiensis var. israelensis in Lab-scale Batch Process, Bioproc. Biosys. Eng. 14(3): 139-144.
20. Liggett, R. W. and Koffler, H. 1948. Corn Steep Liquor in Microbiology. Microbiol. Mol. Biol. Rev., 12: 297.
21. Liu, W. M. and Bajpai, R. K. 1995. A Modified Growth Medium for Bacillus thuringiensis. Biotechnol. Progress., 11: 589-591.
22. Liu, B. L. and Tzeng, Y. M. 2000. Characterization Study of the Sporulation Kinetics of Bacillus thuringiensis. Biotechnol. Bioeng., 68: 11-17.
23. Nazarian Amirani, A., Jahangiri, R., Salehi Jouzani, Gh., Seifinejad, A., Soheilivand, S., Bagheri, O., Keshavarzi, M. and Alamisaeid, K. 2009. Coleopteran-specific and Putative Novel Cry Genes in Iranian Native Bacillus thuringiensis Collection. J. Inverteb. Pathol., 102: 101–109.
24. Ouhib, O., Clavel, T. and Schmitt, P. 2006. The Production of Bacillus cereus Enterotoxins is Influenced by Carbohydrate and Growth Rate. Curr. Microbiol., 53: 222-226.
25. Prabakaran, G. and Hoti, S. L. 2008. Influence of Amino Nitrogen in the Culture Medium Enhances the Production of δ-Endotoxin and Biomass of B. thuringiensis var. israelensis for the Large-scale Production of the Mosquito Control Agent. J. Ind. Microbiol. Biotechnol., 35: 961–965.
26. Prabakaran, G., Hoti, S. L. and Paily, K. P. 2009. Development of Cost-effective Medium for the Large-scale Production of a Mosquito Pupicidal Metabolite from Pseudomonas fluorescens Migula. Biol. Control., 48: 264-266.
27. Rao, Y. K., Tsay, K. J., Wu, W. S. and Tzeng, Y. M. 2007. Medium Optimization of Carbon and Nitrogen Sources for the Production of Spores from Bacillus amyloliquefaciens B128 Using Response Surface Methodology, Process. Biochem., 42: 535-541.
28. Salehi Jouzani, G., Pourjan Abad, A., Seifinejad, A., Marzban, R., Kariman, K. and Maleki, B. 2008a. Distribution and Diversity of Dipteran-specific Cry and Cyt genes in Native Bacillus thuringiensis Strains Obtained from Different Ecosystems of Iran. J. Ind. Microbiol. Biotechnol., 35: 83-94.
29. Salehi Jouzani, G., Seifinejad, A., Saeedizadeh, A., Nazarian, A., Yousefloo, M., Soheilivand, S., Mousivand, M., Jahangiri, R., Yazdani, M., Maali Amiri, R. and Akbari, S. 2008b. Molecular Detection of Nematicidal Crystalliferous Bacillus thuringiensis Strains of Iran and Evaluation of Their Toxicity on Free Living and Plant Parasitic .ematodes. Can. J. Microbiol., 54: 812–822.
30. Seifinejad, A., Salehi Jouzani, G., Hosseinzadeh, A. and Abdmishani, C. 2008. Characterization of Lepidoptera-active Cry and Vip Genes in Iranian Bacillus thuringiensis Strain Collection. J. Biol. Control., 44: 216–226.
31. Shojaaddini, M., Moharramipour, S., Khodabandeh, M. and Talebi, A. A. 2010. Development of a Cost Effective Medium for Production of Bacillus thuringiensis Bioinsecticide Using Food Barley. J. Plant Protec. Res., 50: 9-14.
32. Valicente, F. H. and Mourao, A. H. C. 2008. Use of By-products Rich in Carbon and Nitrogen as a Nutrient Source to Produce Bacillus thuringiensis (Berliner)-based Biopesticide. Neotrop. Entomol., 37: 702-708.
33. Wu, W. T., Hsu, Y. L., Ko, Y. F. and Yao, L. L. 2002. Effect of Shear Stress on Cultivation of Bacillus thuringiensis for Thuringiensin Production. Appl. Microbiol. Biotechnol., 58: 175–177
34. Yezza, A., Tyagi, R. D., Valero, J. R. and Surampalli, R. Y. 2005. Production of Bacillus thuringiensis-based Biopesticides in Batch and Fed Batch Cultures Using Wastewater Sludge as a Raw Material. J. Chem. Technol. Biotechnol., 80: 502–510
35. Zouari, N. and Jaoua, S. 1999. The Effect of Complex Carbon and Nitrogen, Salt, Tween-80 and Acetate on Delta-endotoxin Production by a Bacillus thuringiensis subsp Kurstaki. J. Ind. Microbiol. Biotechnol., 23: 497–502.
36. Zouari, N., Achour, O. and Jaoua S. 2002. Production of Delta-endotoxin by Bacillus thuringiensis subsp Kurstaki and Overcoming of Catabolite Repression by Using Highly Concentrated Gruel and Fish Meal Media in 2- and 20-dm 3 Fermenters. J. Chem. Technol. Biotechnol., 77: 877-882.
Journal of Agricultural Science and Technology
Volume 17, Issue 5
September and October 2015
Pages 1183-1196