Application of Response Surface Methodology for Optimization of Siderophore Production

DOI: 10.18805/IJARe.A-5663    | Article Id: A-5663 | Page : 230-237
Citation :- Application of Response Surface Methodology for Optimization of Siderophore Production.Indian Journal of Agricultural Research.2022.(56):230-237
R.U. Raje Nimbalkar, N.S. Barge, R.J. Marathe, Y.B. Phatake, R.B. Deshmukh, S.S. Dange, S.G. Mane, S.S. Dhawan rjndrmarathe@gmail.com
Address : Department of Microbiology, ADT’s, Shardabai Pawar Mahila Arts, Commerce and Science College, Shardanagar, Baramati-413 115, Maharashtra, India.
Submitted Date : 31-07-2020
Accepted Date : 30-10-2020


Background: In the present study a statistical model (Response Surface Methodology) was proposed for optimization of siderophore production by using Enterobacter hormaechei.
Methods: The rhizospheric soil was used for isolation and isolates were screened for siderophore production by chrome-azurol S (CAS) assay. One potent isolate producing maximum siderophore was selected and characterized by 16S rRNA gene sequencing. The culture conditions were optimized for maximum siderophore production by using Central Composite Design. The response surface curves were used to predict the optimum levels of the factors affecting the yield of siderophore. 
Result: By using rhizospheric soil,eight isolates were obtained and one potent organism was identified as Enterobacter hormaechei subsp. oharae (Accession No. MT 775835) by BLAST. The maximum siderophore production (98%) was obtained in succinate medium and the optimum values of variables were found as pH 7, time 60 hrs, temp. 28°C and succinic acid conc. 0.40%. RSM was used to analyze the data by developing 3D surface plots and the residuals plots. ANOVA was used to determine regression coefficients.


ANOVA Central composite design Chrome-azurol S Enterobacter hormaechei subsp. oharae Regression


  1. Ahmed, E. and Holmström, S.J. (2014). Siderophores in environmental research: roles and applications. Microbial Biotechnology. 7: 196-208.
  2. Akhtar, S. and Ali, B. (2011). Evaluation of rhizobacteria as non-rhizobial inoculants for mung beans. Australian Journal of Crop Science. 5: 1723-1729.
  3. Ali, S.S. and Vidhale, N.N. (2013). Bacterial siderophore and their application: a review. International Journal of Current Microbiology and Applied Sciences. 2: 303-312.
  4. Bisen, P.S. (2014). Microbes in Practice, Edition: First, Chapter: 6 Microbial Staining. IK International, New Delhi. 139-155.
  5. Boukhalfa, H., Lack, J.G., Reilly, S.D., Hersman, L.E. and Neu, M.P. (2003). Siderophore production and facilitated uptake of iron plutonium in P. putida (No. LA-UR-03-0913). Los Alamos National Laboratory. 673: 343-344.
  6. Budzikiewicz, H. (1993). Secondary metabolites from fluorescent Pseudomonads. FEMS, Microbiology Letters. 104: 209-228.
  7. Chantarangsi, W., Liu, W., Bretz, F., Kiatsupaibul, S. and Hayter, A. (2016). Normal Probability Plots with Confidence for the Residuals in Linear Regression. Communications in Statistics - Simulation and Computation. 47: 1-23.
  8. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39: 783-791. 
  9. Gupta, A. and Gopal, M. (2008). Siderophore production by plant growth promoting rhizobacteria. Indian Journal of Agricultural Research. 42(2): 153-156. 
  10. Kannahi, M. and Senbagam, N. (2014). Studies on siderophore production by microbial isolates obtained from rhizosphere soil and its antibacterial activity. Journal of Chemical and Pharmaceutical Research. 6: 1142-1145.
  11. Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution. 16: 111-120.
  12. Kumar, V., Menon, S., Agarwal, H. and Gopalakrishnan, D. (2017). Characterization and optimization of bacterium isolated from soil samples for the production of siderophores. Resource-Efficient Technologies. 3: 434-439.
  13. Kwak, K.O., Jung, S.J., Chung, S.Y., Kan, C.M., Huh, Y.I. and Bae, S.O. (2006). Optimization of culture conditions for CO2 fixation by a chemoautotrophic microorganism, strain YN-1 using factorial design. Biochemical Engineering Journal. 31: 1-7.
  14. Lee, W., Van Baalen, M. and Jansen, V.A. (2012). An evolutionary mechanism for diversity in siderophore producing bacteria. Ecology Letters. 15: 119-125.
  15. Marathe, R.J., Phatake, Y.B. and Sonawane, A.M. (2015). Bio prospecting of Pseudomonas aeruginosa for their potential to produce siderophore, process optimization and evaluation of its bioactivity. International Journal of Bioassays. 4: 3667-3675.
  16. Meyer, J.A. and Abdallah, M.A. (1978). The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. Microbiology. 107: 319-328.
  17. Mokracka, J., Koczura, R. and Kaznowski, A. (2004). Yersinia bactin and other siderophores produced by clinical isolates of Enterobacter spp. and Citrobacter spp. FEMS Immunolgy and Medical Microbiology. 40: 51-55.
  18. Mouafi, F.E., Abo Elsoud, M.M. and Moharam, M.E. (2016). Optimization of biosurfactant production by Bacillus brevis using response surface methodology. Biotechnology Reports. 9: 31-37.
  19. Murugappan, R.M., Aravinth, A., Rajaroobia, R., Karthikeyan, M. and Alamelu, M.R. (2012). Optimization of MM9 medium constituents for enhancement of siderophore genesis in marine Pseudomonas putida using response surface methodology. Indian Journal of Microbiology. 52: 433-441.
  20. Neilands, B. (1987). Universal chemical assay for the detection determination of siderophores. Anal. Biochem. 56: 47-56.
  21. Niehus, R., Picot, A., Oliveira, N.M. and Mitri, S. (2017). Foster, The evolution of siderophore production as a competitive trait. Evolution. 71: 1443-1455.
  22. Pahari, A. and Mishra, B.B. (2017). Characterization of siderophore producing Rhizobacteria and its effect on growth performance of different vegetables. International Journal of Current Microbiology and Applied Sciences. 6: 1398-1405.
  23. Panda, S.H., Goli, J.K., Das, S. and Mohanty, N. (2017). Production, optimization and probiotic characterization of potential lactic acid bacteria producing siderophores. AIMS Microbiology. 3: 88-107.
  24. Pattan, J., Kajale, S. and Pattan, S. (2017). Isolation, production and optimization of siderophores (iron chelators) from Pseudomonas fluorescence NCIM 5096 and Pseudomonas from soil rhizosphere and marine water. International Journal of Current Microbiology and Applied Sciences. 3: 919-928.
  25. Payne, S.M. (1994). Detection, isolation and characterization of siderophores. Methods in Enzymology. 235: 329-344.
  26. Raval, A.A. and Desai, P.B. (2015). Screening and characterization of several siderophore producing bacteria as plant growth-promoters and biocontrolling agents. International Journal of Pharmacy and Life Sciences. 6: 4803-4811.
  27. Saha, M., Sarkar, S., Sarkar, B., Sharma, K., Bhattacharjee, S. and Tribedi, P. (2016). Microbial siderophores and their potential applications: A review. Environmental Science and Pollution Research. 23: 3984-3999.
  28. Saitou, N. and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406-425.
  29. Schwyn, B. and Neilands, J.B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry. 160: 47-56.
  30. Shaikh, S.S., Wani, S.J. and Sayyed, R.Z. (2016). Statistical-based optimization and scale-up of siderophore production process on laboratory bioreactor. Biotech. 6: 69.
  31. Shen, C. and Zhang, Y. (2017). Staining technology and bright-field microscope use in food microbiology laboratory for the food science student. Springer. Cham. 9-14.
  32. Silva-Stenico, M.E., Pacheco, F.T.H., Rodrigues, J.L.M., Carrilho, E. and Tsai, S. (2005). Growth and siderophore production of Xylella fastidiosa under iron-limited conditions. Microbiological Research. 160: 429-436.
  33. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution. 30: 2725-2729.
  34. Tanyildizi, M.S., Özer, D. and Elibol, M. (2005). Optimization of α-amylase production by Bacillus sp. using response surface methodology. Process Biochemistry. 40: 2291-    2296.
  35. Troeh, F.R. and Thompson, L.M. (2005). Soils and soil fertility. New York, USA: Blackwell. 489.

Global Footprints