Production and Characterization of Lipopeptide Biosurfactant from Bacillus subtilis A8-8

  • Lee Sang-Cheol (Department of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University) ;
  • Yoo Ju-Soon (Department of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University) ;
  • Kim Sun-Hee (Department of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University) ;
  • Chung Soo-Yeol (Department of Food Science, Dong-Ju College) ;
  • Hwang Cher-Won (Institute of Bioscience and Technology, Han-Dong Global University) ;
  • Joo Woo-Hong (Department of Biology, Chang-Won National University) ;
  • Choi Yong-Lark (Department of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University)
  • Published : 2006.05.01

Abstract

A biosurfactant-producing bacterial strain was selected from oil-contaminated soil because of its ability to degrade crude oil and tributyrin $(C_{4:0})$. The strain was identified as Bacillus subtilis A8-8 based on its morphological, biochemical, and physiological characteristics. When B. subtilis A8-8 was grown with crude oil as the sole carbon source, the biosurfactant from the strain emulsified crude oil, vegetable oil, and hydrocarbons. Soybean oil was the optimum substrate for the emulsifying activity and emulsion stability of the biosurfactant, both of which were superior to those of several commercially available surfactants. The biosurfactant was purified by a procedure including HCl precipitation, methanol treatment, and silica-gel chromatography. The partially purified biosurfactant was analyzed by TLC (thin-layer chromatography), SDS-PAGE, and HPLC and it reduced the surface tension of water from 72 mN/m to 26 mN/m at a concentration of 30 mg/l. Therefore, the purified lipopeptide biosurfactant has strong properties as an emulsifying agent and acts as an emulsion-stabilizing agent.

Keywords

References

  1. Arima, K., A. Kakinuma, and G. Tamura. 1968. Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: Isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31: 488-494 https://doi.org/10.1016/0006-291X(68)90503-2
  2. Baek, K. H., H. S. Kim, S. H. Moon, I. S. Lee, H. M. Oh, and B. D. Yoon. 2004. Effects of soil types on the biodegradation of crude oil by Nocardia sp. H17-1. J. Microbiol. Biotechnol. 14: 901-905 https://doi.org/10.1159/000076921
  3. Banat, I. M. 1995. Characterization of biosurfactants and their use in pollution removal-state of the art. Acta Biotechnol. 15: 251-267 https://doi.org/10.1002/abio.370150302
  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  5. Bechard, J., K. C. Eastwell, P. L. Sholber, G. Mazza, and B. Skura. 1998. Isolation and partial chemical characterization of an antimicrobial peptide produced by a strain of Bacillus subtilis. J. Agric. Food Chem. 46: 5355-5361 https://doi.org/10.1021/jf9803987
  6. Cha, J. Y., B. G. Kim, S. Y. Chung, Y. S. Cho, Y. L. Choi, and Y. L. Lee. 1999. Characterization of crude oil degradation by Klebsiella sp. KCL-1 isolated from sea water. Kor. J. Appl. Microbiol. Biotechnol. 27: 452-457
  7. Cho, W. S., E. H. Lee, E. H. Shim, J. S. Kim, H. W. Ryu, and K. S. Cho. 2005. Bacterial communities of biofilms sampled from seepage groundwater contaminated with petroleum oil. J. Microbiol. Biotechnol. 15: 952-964
  8. Chun, J. and K. S. Bae. 2000. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence. Antonie van Leeuwenhoek 78: 123-127 https://doi.org/10.1023/A:1026555830014
  9. Cirigliano, M. C. and G. M. Carman. 1984. Isolation of a bioemulsifier from Candida lipolytica. Appl. Environ. Microbiol. 48: 747-750
  10. Cirigliano, M. C. and G. M. Carman. 1985. Purification and characterization of liposan, a bioemulsifier from Candida lipolytica. Appl. Environ. Microbiol. 50: 846-850
  11. Desai, J. D. and I. M. Banat. 1997. Microbial production of surfactants and their commercial potential. Microbiol. Molecul. Biol. Rev. 61: 47-64
  12. Eppard, M., W. E. Krrumbein, C. Kock, E. Rhiel, J. T. Sraley, and E. Stackebrandt. 1996. Morphological, physiological, and molecular characterization of actinomycetes isolated from dry soil, rocks, and monuments surfaces. Arch. Microbiol. 166: 12-22 https://doi.org/10.1007/s002030050350
  13. Georgiou, G., S. C. Lim, and M. M. Sharma. 1992. Surfaceactive compounds from microorganisms. Bio/technology 10: 60-65 https://doi.org/10.1038/nbt0192-60
  14. Gerhardt, P., R. G. E. Murray, R. N. Costilow, and E. W. Wester. 1981. Manual of Methods for General Bacteriology. American Society for Microbiology, N.Y., U.S.A
  15. Gray, J. P. and R. P. Herwig. 1996. Phylogenetic analysis of the bacterial of the bacterial communities in marine sediments. Appl. Environ. Microbiol. 62: 4049-4059
  16. Greek, B. F. 1991. Sales of detergents growing despite recession. Chem. Eng. News 69: 25-52
  17. Huang, C. C., A. Takashi, and S. Makoto. 1993. Nucleotide sequence and characteristics of the gene, lpa-14, responsible for biosynthesis of the lipopeptide antibiotics iturin A and surfactin from Bacillus subtilis RB14. J. Ferment. Bioengin. 76: 445-450 https://doi.org/10.1016/0922-338X(93)90238-4
  18. Hourdou, M. L. and F. Besson. 1994. Surfactin et antibiotiques ituriniques: Structure proriet'es et biosynthese des lipopeptides de Bacillus subtilis. Regard. Biochim. 1: 35-42
  19. John, G. H., N. R. Krieg, and P. H. A. Sneath. 1994. Bergey's Manual of Systematic Bacteriology. 9th Ed. Williams and Wilkins, Baltimore
  20. Kakinuma, A., A. Ouchida, T. Shima, H. Sugino, M. Isono, G. Tamura, and K. Arima. 1969. Conformation of the structure of surfactin by mass spectrometry. Agric. Biol. Chem. 33: 1669-1671 https://doi.org/10.1271/bbb1961.33.1669
  21. Kalinovskaya, N. I., T. A. Kuznetsova, E. P. Ivanova, L. A. Romanenko, V. G. Voinov, F. Huth, and H. Laatsch. 2002. Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from Ascidian Halocynthia aurantium. Mar. Biotechnol. (NY) 4: 179-188 https://doi.org/10.1007/s10126-001-0084-4
  22. Katz, E. and A. L. Demain. 1977. The peptide antibiotics of Bacillus: Chemistry, biogenesis and possible functions. Bacteriol. Rev. 41: 449-474
  23. Kim, S. H., B. D. Yoon, C. H. Lee, H. Y. Seo, H. M. Oh, T. Katsuragi, and Y. Tani. 1997. Production and properties of a lipopeptide biosurfactant from Bacillus subtilis C9. J. Ferment. Bioengin. 28: 71-75
  24. Kim, S. H., E. J. Lim, S. O. Lee, J. D. Lee, and T. H. Lee. 2000. Purification and characterization of biosurfactants from Nocardia sp. L-417. Biotech. Appl. Biochem. 31: 249-253 https://doi.org/10.1042/BA19990111
  25. Kim, S. H. and K. T. Jang. 2001. Protein Experiment Note. Revised Ed. World Science Press
  26. Kim, S. W., J. S. Yoo, S. C. Lee, Y. S. Cho, Y. C. Lee, and Y. L. Choi. 2001. Isolation of oil-degrading bacterium, Providencia rettgeri 4A3 and characterization of crude oil degradation. Kor. J. Life Sci. 11: 530-536
  27. Kretschner, A., H. Bock, and F. Wagnee. 1982. Chemical and physical characterization of interfacial-active lipids from Rhodococcus erythropolis grown on n-alkane. Appl. Environ. Microbiol. 44: 864-870
  28. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, and A. Danchin, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249-256 https://doi.org/10.1038/36786
  29. Kuske, C. R., S. M. Bams, and J. D. Busch. 1997. Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions. Appl. Environ. Microbiol. 63: 3614-3621
  30. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage $T_4$. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  31. Lee, S. C., Y. J. Jung, J. S. Yoo, Y. S. Cho, I. H. Cha, and Y. L. Choi. 2002. Characteristic of biosurfactants produced by Bacillus sp. LSC11. Kor. J. Life Sci. 12: 745-751 https://doi.org/10.5352/JLS.2002.12.6.745
  32. Magaritis, A., K. Kennedy, J. E. Zajic, and D. F. Gerson. 1979. Biosurfactant production by Nocardia erythropolis. Dev. Ind. Microbiol. 20: 623-630
  33. Morikawa, M., Y. Hirata, and T. Imanaka. 2000. A study on the structure-function relationship of lipopeptide biosurfactants. Biochem. Biophys. Acta 1488: 211-218 https://doi.org/10.1016/S1388-1981(00)00124-4
  34. Parkinson, M. 1985. Bio-surfactant. Biotechnol. Adv. 3: 65-83 https://doi.org/10.1016/0734-9750(85)90006-0
  35. Reva, O. N., C. Dixelius, J. Meijer, and F. G. Priest. 2004. Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol. Ecol. 48: 249-259 https://doi.org/10.1016/j.femsec.2004.02.003
  36. Ron, E. Z. and E. Rosenberg. 2001. Natural roles of biosurfactant. Environ. Microbiol. 3: 229-236 https://doi.org/10.1046/j.1462-2920.2001.00190.x
  37. Rosenberg, E. and E. Z. Ron. 1999. High- and low-molecularmass microbial surfactants. Appl. Microbiol. Biotechnol. 52: 154-162 https://doi.org/10.1007/s002530051502
  38. Siefert, J. L., M. Larios-Sanz, L. K. Nakamura, R. A. Slepecky, J. H. Paul, E. R. Moore, G. E. Fox, and P. Jurtshuk, Jr. 2000. Phylogeny of marine Bacillus isolates from the Gulf of Mexico. Curr. Microbiol. 41: 84-88 https://doi.org/10.1007/s002840010098
  39. Suk, W. S., H. J. Son, G. Lee, and S. J. Lee. 1999. Purification and characterization of biosurfactants produced by Pseudomonas sp. SW 1. J. Microbiol. Biotechnol. 9: 56-61
  40. Symmank, H., P. Franke, W. Saenger, and F. Bernhard. 2002. Modification of biologically active peptides: Production of a novel lipopeptide after engineering of Bacillus subtilis surfactin synthetase. Protein Engin. 15: 913-921 https://doi.org/10.1093/protein/15.11.913
  41. Touchstone, J. C. 1992. Practice of Thin Layer Chromatography. pp. 166. 3rd. Ed. John Wiley & Sons, Inc
  42. Wise, W. G., J. V. McArthur, and L. J. Skimkets. 1997. Bacterial diversity of a Carolima bay as determined by 16S rRNA gene analysis: Confirmation of novel taxa. Appl. Environ. Microbiol. 63: 1505-1514
  43. Woo, S. H. and J. M. Park. 2004. Biodegradation of aromatic compound from soil by drum bioreactor system. J. Microbiol. Biotechnol. 14: 435-441
  44. Yakimov, M. M., K. N. Timmis, V. Wray, and H. L. Fredrickson. 1995. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl. Environ. Microbiol. 61: 1706-1713
  45. Yoo, D. S., B. S. Lee, and E. K. Kim. 2005. Characteristics of microbial biosurfactant as an antifungal agent against plant pathogenic fungus. J. Microbiol. Biotechnol. 15: 1164-1169
  46. Zajic, J. E., H. Guignard, and D. F. Gerson. 1977. Properties and biodegradation of a bioemulsifier from Corynebacterium hydrocarboclastus. Biotechnol. Bioeng. 19: 1303-1320 https://doi.org/10.1002/bit.260190905