Optimization of Medium Composition for Lipopeptide Production from Bacillus subtilis N7 using Response Surface Methodology

  • Luo, Yi (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University) ;
  • Zhang, Guoyi (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University) ;
  • Zhu, Zhen (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University) ;
  • Wang, Xiaohui (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University) ;
  • Ran, Wei (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University) ;
  • Shen, Qirong (Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University)
  • Received : 2012.07.23
  • Accepted : 2012.11.01
  • Published : 2013.03.28


The nutritional requirements for the maximum production of lipopeptides by Bacillus subtilis N7 (B. subtilis N7) were investigated and optimized using response surface methodology (RSM) under shake flask fermentation. A one-factor-at-a-time experimental setup was used to screen carbon and nitrogen sources. A Plackett-Burman design (PBD) was employed to screen the most critical variables for lipopeptides production amongst ten nutritional elements. The central composite experimental design (CCD) was finally adopted to elucidate the composition of the fermentation medium. Statistical analyses (analysis of variance, ANOVA) of the results showed that KCl, $MnSO_4$ and $FeSO_4{\cdot}6H_2O$ were important components and that their interactions were strong. Lipopeptide production was predicted to reach 709.87 mg/L after a 60 h incubation using an optimum fermentation medium composed of glucose 7.5 g/L, peanut oil 1.25 g/L, $MgSO_4$ 0.37 g/L, $KH_2PO_4$ 0.75 g/L, monosodium glutamate 6.75 g/L, yeast extract and $NH_4Cl$ (5:3 w/w) 10 g/L, KCl 0.16 g/L, $FeSO_4{\cdot}6H_2O$ 0.24 mg/L, $MnSO_4$ 0.76 mg/L, and an initial pH of 7.0. Lipopeptide production ($706.57{\pm}3.70$ mg/L) in the optimized medium confirmed the validity of the predicted model.


Lipopeptides;Bacillus subtilis;optimization;response surface methodology


  1. Amanullah, A., C. McFarlane, A. Emery, and A. Nienow. 2001. Scale - down model to simulate spatial pH variations in large - scale bioreactors. Biotechnol. Bioeng. 73: 390-399.
  2. Asaka, O. and M. Shoda. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62: 4081-4085.
  3. Bernheimer, A. and L. S. Avigad. 1970. Nature and properties of a cytolytic agent produced by Bacillus subtilis. J. Gen. Microbiol. 61: 361.
  4. Chen, H. C. 1996. Optimizing the concentrations of carbon, nitrogen and phosphorus in a citric acid fermentation with response surface method. Food Biotechnol. 10: 13-27.
  5. Cooper, D. G. and J. D. Sheppard. 1991. The response of Bacillus subtilis ATCC 21332 to manganese during continuous- phased growth. Appl. Microbiol. Biotechnol. 35: 72-76.
  6. Finking, R. and M. A. Marahiel. 2004. Biosynthesis of nonribosomal peptides 1. Annu. Rev. Microbiol. 58: 453-488.
  7. Heerklotz, H. and J. Seelig. 2007. Leakage and lysis of lipid membranes induced by the lipopeptide surfactin. Eur. Biophysics J. 36: 305-314.
  8. Hutadilok-Towatana, N., A. Painupong, and P. Suntinanalert. 1999. Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719. J. Biosci. Bioeng. 87: 581-587.
  9. Marks, E. 1968. Profile analysis in a two-way classification problem. Multivar. Behav. Res. 3: 95-106.
  10. Miethke, M., H. Westers, E. J. Blom, O. P. Kuipers, and M. A. Marahiel. 2006. Iron starvation triggers the stringent response and induces amino acid biosynthesis for bacillibactin production in Bacillus subtilis. J. Bacteriol. 188: 8655-8657.
  11. Mizumoto, S., M. Hirai, and M. Shoda. 2006. Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation. Appl. Microbiol. Biotechnol. 72: 869-875.
  12. Ongena, M. and P. Jacques. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trend. Microbiol. 16: 115-125.
  13. Pareek, N., R. P. Singh, and S. Ghosh. 2011. Optimization of medium composition for enhanced chitin deacetylase production by mutant Penicillium oxalicum SAE (M)-51 using response surface methodology under submerged fermentation. Process Biochem. 46: 1693-1697.
  14. Quentin, M., F. Besson, F. Peypoux, and G. Michel. 1982. Action of peptidolipidic antibiotics of the iturin group on erythrocytes: Effect of some lipids on hemolysis. Biochimica et Biophysica Acta (BBA)-Biomembranes. 684: 207-211.
  15. Rado, T. A. and J. A. Hoch. 1973. Phosphotransacetylase from Bacillus subtilis: purification and physiological studies. Biochimica et Biophysica Acta (BBA)-Enzymology. 321: 114-125.
  16. Shih, I.-L., C.-Y. Lin, J.-Y. Wu, and C. Hsieh. 2009. Production of antifungal lipopeptide from Bacillus subtilis in submerged fermentation using shake flask and fermentor. Korean J. Chem. Eng. 26: 1652-1661.
  17. Stein, T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56: 845-857.
  18. Ujita, S. and K. Kimura. 1982. Glucose-6-phosphate dehydrogenase, vegetative and spore Bacillus subtilis. Methods in Enzymol. 89: 258-261.
  19. Wei, Y. H., L. F. Wang, and J. S. Chang. 2004. Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis. Biotechnol. Progr. 20: 979-983.
  20. Wu, A.-L., T. Chen, Y. Gan, X. Chen, and X.-M. Zhao. 2007. Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Appl. Microbiol. Biotechnol. 76: 783-794.