Food and Bioprocess Technology

, Volume 4, Issue 5, pp 745–752 | Cite as

Flocculation Properties of Poly(γ-Glutamic Acid) Produced from Bacillus subtilis Isolate

Original Paper


Bacillus subtilis R 23 produced extracellular biopolymer showing excellent flocculation activity. The biopolymer was confirmed as poly(γ-glutamic acid) (PGA) using high-performance liquid chromatography profile and product characterization. The production, characteristics, and flocculation properties of PGA were studied. PGA produced by B. subtilis R 23 was devoid of any polysaccharides and had a molecular weight of 6.2 × 106 Da. The flocculating activity of PGA in the kaolin suspension was markedly stimulated by the addition of bivalent and trivalent cations in optimum concentration. The pH of reaction mixture also influenced the flocculating activity. Response surface methodology was used to establish the optimum parameters for maximum flocculating activity and to study their interactions. A maximum flocculating activity of 30.32 ± 1.4 1/optical density was obtained with 7.5 mg/L of PGA in combination with 8.0 mM of Ca+2 at pH 7.5.


Poly(γ-glutamic acid) Bacillus subtilis Flocculating activity Bioflocculant Marine bacteria 


  1. Ashiuchi, M., Kamei, T., Baek, D. H., Shin, S. Y., Sung, M. H., Soda, K., et al. (2001). Isolation of Bacillus subtilis (chungkookjang), a poly-γ-glutamate producer with high genetic competence. Applied Microbiology and Biotechnology, 57, 764–769. doi:10.1007/s00253-001-0848-9.CrossRefGoogle Scholar
  2. Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Optimization of Aspergillus niger fermentation for the production of glucose oxidase. Food and Bioprocess Technology. doi:10.1007/s11947-007-0050-x.
  3. Chen, X., Chen, S., Sun, M., & Yu, Z. (2005). Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate. Applied Microbiology and Biotechnology, 69, 390–396. doi:10.1007/s00253-005-1989-z.CrossRefGoogle Scholar
  4. Cromwick, A. M., Birrer, G. A., & Gross, R. A. (1996). Effects of pH and aeration on γ-poly(glutamic acid) formation by Bacillus licheniformis in controlled batch fermentor cultures. Biotechnology and Bioengineering, 50, 222–227. doi:10.1002/(SICI)1097-0290(19960420)50:2<222::AID-BIT10>3.0.CO;2-P.CrossRefGoogle Scholar
  5. Deng, S. B., Bai, R. B., Hu, X. M., & Luo, Q. (2003). Characteristics of a bioflocculants produced by Bacillus mucilaginosus and its use in starch wastewater treatment. Agricultural and Biological Chemistry, 60, 588–593.Google Scholar
  6. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric methods for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. doi:10.1021/ac60111a017.CrossRefGoogle Scholar
  7. Gong, W. X., Wang, S. G., Sun, X. F., Liu, X. W., Yue, Q. Y., & Gao, B. Y. (2008). Bioflocculant production by culture of Serratia ficaria and its application in wastewater treatment. Bioresource Technology, 99, 4668–4674. doi:10.1016/j.biortech.2007.09.077.CrossRefGoogle Scholar
  8. Goto, A., & Kunioka, M. (1992). Biosynthesis and hydrolysis of poly (γ-glutamic acid) from Bacillus subtilis IFO3335. Bioscience, Biotechnology, and Biochemistry, 56, 1031–1035.CrossRefGoogle Scholar
  9. Hezayen, F. F., Rehm, B. H., Tindall, B. J., Steinbuchel, A., & Eberhardt, R. (2001). Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. nov. and description of Natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid). International Journal of Systematic and Evolutionary Microbiology, 51, 1133–1142.Google Scholar
  10. Houghton, J. I., & Quarmby, J. (1999). Biopolymers in wastewater treatment. Current Opinion in Biotechnology, 10, 259–262. doi:10.1016/S0958-1669(99)80045-7.CrossRefGoogle Scholar
  11. Kunioka, M., & Goto, A. (1994). Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid, citric acid, and ammonium sulfate in Bacillus subtilis IFO3335. Applied Microbiology and Biotechnology, 40, 867–872. doi:10.1007/BF00173990.CrossRefGoogle Scholar
  12. Kurane, R., Toeds, K., Takeda, K., & Suzuki, T. (1986). Culture conditions for production of microbial flocculant by Rhodococcus erythropolis. Agricultural and Biological Chemistry, 50, 2309–2313.Google Scholar
  13. Li, Y., He, N., Guan, H., Du, G., & Chen, J. (1999). A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: A proposed biosynthetic pathway and experimental confirmation. Applied Microbiology and Biotechnology, 52, 698–703. doi:10.1007/s002530051581.CrossRefGoogle Scholar
  14. Lian, B., Chen, Y., Zhao, J., Teng, H., Zhu, L., & Yuan, S. (2008). Microbial flocculation by Bacillus mucilaginosus: Applications and mechanisms. Bioresource Technology, 99, 4825–4831. doi:10.1016/j.biortech.2007.09.045.CrossRefGoogle Scholar
  15. Mahmoud, D. A. R. (2006). Isolation of polyglutamic acid flocculant producing bacteria from extreme Egyptian environments. Journal Of Applied Sciences Research, 2(9), 608–612.Google Scholar
  16. Mamatha, S. S., Ravi, R., & Venkateswaran, G. (2008). Medium optimization of gamma linolenic aid production in Mucor rouxii CFR-G15 using RSM. Food Bioprocess Technology, 1, 405–409. doi:10.1007/s11947-008-0103-9.CrossRefGoogle Scholar
  17. Salehizadeh, H., Vossoughi, M., & Alemzadeh, I. (2000). Some investigations on bioflocculant producing bacteria. Biochemical Engineering Journal, 5, 39–44. doi:10.1016/S1369-703X(99)00066-2.CrossRefGoogle Scholar
  18. Shih, I. L., & Van, Y. T. (2001). The production of poly(γ-glutamic acid) from microorganism and its various applications. Bioresource Technology, 79, 207–225. doi:10.1016/S0960-8524(01)00074-8.CrossRefGoogle Scholar
  19. Shih, I. L., Van, Y. T., Yeh, L. C., Lin, H. G., & Chang, Y. N. (2001). Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresource Technology, 78, 267–272. doi:10.1016/S0960-8524(01)00027-X.CrossRefGoogle Scholar
  20. Shih, I. L., Van, Y. T., & Chang, Y. N. (2002). Application of statistical experimental methods to optimize production of poly(γ-glutamic acid) by Bacillus licheniformis CCRC 12826. Enzyme and Microbial Technology, 31, 213–220. doi:10.1016/S0141-0229(02)00103-5.CrossRefGoogle Scholar
  21. Suh, H. H., Kwon, G. S., Lee, C. H., Kim, H. S., Oh, H. M., & Yoon, B. D. (1997). Characterization of bioflocculant produced by Bacillus sp. DP-152. Journal of Fermentation and Bioengineering, 84, 108–112. doi:10.1016/S0922-338X(97)82537-8.CrossRefGoogle Scholar
  22. Suo, C., Mei, L. H., Huang, J., & Sheng, Q. (2007). Selection of γ-poly glutamic acid high yield strain by 60Co γ-irradiation and the optimization of its culture medium. Journal of chemical and engineering of Chinese Universities, 21, 820–825.Google Scholar
  23. Takeda, M., Koizumi, J. I., Matsuoka, H., & Hikuma, M. (1992). Factors affecting the activity of a protein bioflocculant produced by Nocardia amarae. Journal of Fermentation and Bioengineering, 74, 408–409. doi:10.1016/0922-338X(92)90043-T.CrossRefGoogle Scholar
  24. Toeda, K., & Kurane, R. (1991). Microbial flocculant from Alcaligenes cupidus KT 201. Agricultural and Biological Chemistry, 55, 2793–2799.Google Scholar
  25. Wu, J. Y., & Ye, H. F. (2007). Characterization and flocculating properties of an extracellular biopolymer produced from a Bacillus subtilis DYU1 isolate. Process Biochemistry, 42, 1114–1123. doi:10.1016/j.procbio.2007.05.006.CrossRefGoogle Scholar
  26. Yokoi, H., Natsuda, O., Hirose, J., Hayashi, S., & Takasaki, Y. (1995). Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90. Journal of Fermentation and Bioengineering, 79, 378–380. doi:10.1016/0922-338X(95)94000-H.CrossRefGoogle Scholar
  27. Yokoi, H., Arima, T., Hirose, J., Hayashi, S., & Takasaki, Y. (1996). Flocculation properties of poly(gamma-glutamic acid) produced by Bacillus subtilis. Journal of Fermentation and Bioengineering, 82, 84–87. doi:10.1016/0922-338X(96)89461-X.CrossRefGoogle Scholar

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© Springer Science + Business Media, LLC 2009

Authors and Affiliations

  1. 1.Food Engineering and Technology Department, Institute of Chemical TechnologyUniversity of MumbaiMumbaiIndia

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