Evaluation of various methods of selection of B. subtilis strains capable of secreting surface-active compounds

Autoři: Beata Koim-Puchowska aff001;  Grzegorz Kłosowski aff001;  Dawid Mikulski aff001;  Aleksandra Menka aff001
Působiště autorů: Kazimierz Wielki University, Department of Biotechnology, Bydgoszcz, Poland aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225108


The aim of the study was the evaluation of a three-step method for the selection of bacterial strains capable of producing surfactin. The procedure consisted of the following steps: 1.blood agar test, 2. measurement of the surface tension (ST) of the medium using the du Nouy method before and after submerged culture, 3. qualitative and quantitative assessment of surfactin by HPLC. Forty five Bacillus subtilis natto strains producing haemolysis zones (≥3mm) were selected. Nineten of them reduced ST of the medium to ≤ 40 mN/m; in six cases, the reduction was as much as 50%. All indicated strains produced surfactin. Positive correlations (p <0.5) between the percentage reduction of ST of the medium and surfactin concentration (r = 0.44), indicate that this parameter is determinant of the ability to synthesize this compound. The blood agar test has been shown to be useful only as a pre-selection criterion for surfactin producers (18 strains selected by this method reduced ST by only ≤30%). The proposed selection strategy proved effective and made it possible to select the BS15 strain that reduced the ST of the medium to 30.56 ± 0.15 mN/m and simultaneously provided a high concentration of surfactin compared to other strains.

Klíčová slova:

Bacillus subtilis – Biosynthesis – Blood – High performance liquid chromatography – Mechanical tension – Sheep – Surface tension – Surfactants


1. Knepper TP, Berna JL, Surfactants: Properties, production, and environmental Aspects, Analysis and Fate of Surfactants and the Aquatic Environment in: Comprehensive Analytical Chemistry 2003, pp. 1–49.

2. Acmite Market Intelligence Market Report: World Surfactant Market. Archived from the original on April 2016. 4th edition.

3. Banat IM, Makkar RS, Cameotra SS. Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol. 2000; 53: 495–508. doi: 10.1007/s002530051648 10855707

4. Ivanković T, Hrenović J. Surfactants in the environment. Arh Hig Rada Toksikol. 2010; 61: 95–110. doi: 10.2478/10004-1254-61-2010-1943 20338873

5. Jackson SA, Borchert E, O’Gara F, Dobson ADW. Metagenomics for the discovery of novel biosurfactants of environmental interest from marine ecosystems. Curr Opin Biotechnol. 2015; 33: 176–182. doi: 10.1016/j.copbio.2015.03.004 25812477

6. Shin K-H, Kim K-W, Ahn Y. Use of biosurfactant to remediate phenanthrene-contaminated soil by the combined solubilization-biodegradation process. J Hazard Mate. 2006; 137: 1831–1837.

7. Mukherjee S, Das P, Sen R. Towards commercial production of microbial surfactants. Trends Biotechnol 2006; 24: 509–515. doi: 10.1016/j.tibtech.2006.09.005 16997405

8. Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NA-H. Characterization of surfactin produced by Bacillus subtilis isolate BS5. Appl Biochem Biotechnol. 2008; 150: 289–303. doi: 10.1007/s12010-008-8153-z 18437297

9. Al-Bahry SN, Al-Wahaibi YM, Elshafie AE, Al-Bemani AS, Joshi SJ, Al-Makhmari HS, et al. Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery. Int Biodeter Biodegr. 2016; 81: 141–146.

10. Gudiña EJ, Fernandes EC, Rodrigues AI, Teixeira JA, Rodrigues LR. Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium. Front Microbiol. 2015; 6: 59. doi: 10.3389/fmicb.2015.00059 25705209

11. Santos DKF, Rufino RD, Luna JM, Santos VA, Sarubbo LA. Biosurfactants: Multifunctional Biomolecules of the 21st Century. Int J Mol Sci 2016; 17: 401. doi: 10.3390/ijms17030401 26999123

12. Das K, Mukherjee AK. Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: Some industrial applications of biosurfactants. Process Biochem. 2007; 42: 1191–1199.

13. Wei Y-H, Wang L-Ch, Chen W-Ch, Chen S-Y. Production and characterization of fengycin by indigenous Bacillus subtilis F29-3 originating from a potato farm. Int J Mol Sci. 2010; 11: 4526–4538. doi: 10.3390/ijms11114526 21151454

14. Gudiña EJ, Rangarajan V, Sen R, Rodrigues LR. Potential therapeutic applications of biosurfactants. Trends Pharmacol Sci. 2013; 34: 667–675. doi: 10.1016/j.tips.2013.10.002 24182625

15. Banat IM, Satpute SK, Cameotra SS, Patil R, Nyayanit NV. Cost effective technologies and renewable substrates for biosurfactants’ production. Front Microbiol. 2014; 5: 697. doi: 10.3389/fmicb.2014.00697 25566213

16. Sobrinho HB, Luna JM, Rufino RD, Porto ALF, Sarubbo LA. Biosurfactants: classification, properties and environmental applications. Biotechnology 2014; 11: 1–29.

17. Chaprão MJ, Ferreira INS, Correa PF, Rufino RD, Luna JM, Silva EJ, et al. Application of bacterial and yeast biosurfactants for enhanced removal and biodegradation of motor oil from contaminated sand. Electron J Biotechn. 2015; 18: 471–479.

18. Bonmatin J-M, Laprevote O, Peypoux F. Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents. Comb Chem High T Scr. 2003; 6: 541–556.

19. Jacques P. Surfactin and other lipopeptides from Bacillus spp. In Biosurfactants. Microbiology Monographs; Soberón-Chávez G. Springer, Berlin, Heidelberg; 2011; pp.57–91.

20. Chen W-Ch, Juang R-S, Wei Y-H. Applications of a lipopeptide biosurfactant, surfactin, produced by microorganisms. Biochem Eng J. 2015; 103: 158–169.

21. Willenbacher J, Yeremchuk W, Mohr T, Syldatk Ch, Hausmann R. Enhancement of surfactin yield by improving the medium composition and fermentation process. AMB Express 2015; 5: 57.

22. Vollenbroich D, Özel M, Vater J, Kamp RM, Pauli G. Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals 1997; 25: 289–297. doi: 10.1006/biol.1997.0099 9324997

23. Wu Y-S, Ngai S-C, Goh B-H, Chan K-G, Lee L-H, Chuah L-H. Anticancer activities of surfactin and potential application of nanotechnology assisted surfactin delivery. Front Pharmacol. 2017; 8: 761. doi: 10.3389/fphar.2017.00761 29123482

24. Lai CC, Huang YC, Wei YH, Chang JS. Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil. J Hazard Mater. 2009; 167: 609–614. doi: 10.1016/j.jhazmat.2009.01.017 19217712

25. Peypoux F, Bonmatin J, Wallach J. Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol. 1999; 51: 553–563. doi: 10.1007/s002530051432 10390813

26. Liu J-F, Mbadinga SM, Yang S-Z, Gu J-D, Mu B-Z. Chemical structure, property and potential applications of biosurfactants produced by Bacillus subtilis in petroleum recovery and spill mitigation. Int J Mol Sci 2015; 16: 4814–4837. doi: 10.3390/ijms16034814 25741767

27. Sen R, Swaminathan T. Characterization of concentration and purification parameters and operating conditions for the small-scale recovery of surfactin. Process Biochem. 2005; 40: 2953–2958.

28. Vedaraman N, Venkatesh N. Production of surfactin by Bacillus subtilis MTCC 2423 from waste frying oils. Braz J Chem Eng. 2011; 28: 175–180.

29. Willenbacher J, Zwick M, Mohr T, Schmid F, Syldatk C, Hausmann R. Evaluation of different Bacillus strains in respect of their ability to produce surfactin in a model fermentation process with integrated foam fractionation. Appl Microbiol Biotechnol. 2014; 98: 9623–9632. doi: 10.1007/s00253-014-6010-2 25158834

30. Nitschke M, Pastore GM. Biosurfactant production by Bacillus subtilis using cassava-processing effluent. Appl Biochem Biotechnol. 2004; 112: 163–172. doi: 10.1385/abab:112:3:163 15007184

31. Zhao F, Shi R, Cui Q, Han S, Dong H, Zhang Y. Biosurfactant production under diverse conditions by two kinds of biosurfactant-producing bacteria for microbial enhanced oil recovery. J Pet Sci Eng. 2017; 157: 124–130.

32. Mubarak MQE, Hassan AR, Hamid AA,. Khalil S, Isa MHM. A simple and effective isocratic HPLC method for fast identification and quantification of surfactin. Sains Malays. 2015; 44: 115–120.

33. Czaczyk K, Marciniak A, Białas W, Mueller A, Myszka K. The effect of environmental factors influencing lipopeptide biosurfactants biosynthesis by Bacillus spp. Food. Science. Technology. Quality 2007; 1: 140–149.

34. Zhu Z, Zhang F, Wei Z, Ran W, Shen Q. The usage of rice straw as a major substrate for the production of surfactin by Bacillus amyloliquefaciens XZ-173 in solid-state fermentation. J Environ Manage. 2013; 127: 30, 96–102.

35. Pathak KV, Keharia H. Application of extracellular lipopeptide biosurfactant produced by endophytic Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) in microbially enhanced oil recovery (MEOR). 3 Biotech. 2014; 4: 41–48. doi: 10.1007/s13205-013-0119-3 28324457

36. Gurjar J, Sengupta B. Production of surfactin from rice mill polishing residue by submerged fermentation using Bacillus subtilis MTCC 2423. Bioresour Technol. 2015; 189: 243–249. doi: 10.1016/j.biortech.2015.04.013 25898085

37. Alonso S, Martin PJ. Impact of foaming on surfactin production by Bacillus subtilis: implications on the development of integrated in situ foam fractionation removal systems. Biochem Eng J. 2016; 110: 125–133.

38. Taskin M, Kurbanoglu EB. Evaluation of waste chicken feathers as peptone source for bacterial growth. J Appl Microbiol. 2011; 111: 4, 826–834. doi: 10.1111/j.1365-2672.2011.05103.x 21762471

39. Slivinsky CT, Mallmann E, de Araújo JM, Mitchell DA, Krieger N. Production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation using a medium based on okara with sugarcane bagasse as a bulking agent. Process Biochem. 2012; 47: 1848–1855.

40. Jajor P, Piłakowska-Pietras D, Krasowska A, Łukaszewicz M. Surfactin analogues produced by Bacillus subtilis strains grown on rapeseed cake. J Mol Struct. 2016; 1126: 141–146.

41. Ratledge C, Kristiansen B. Basic Biotechnology. Third edition. Cambridge University Press 2006. doi: 10.1186/1472-6750-6-31 16820068

42. Nitschke M, Pastore GM. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresour Technol. 2006; 97: 336–341. doi: 10.1016/j.biortech.2005.02.044 16171690

43. Joshi S, Bharucha C, Jha S, Yadav S, Nerurkar A, Desai AJ. Biosurfactant production using molasses and whey under thermophilic conditions. Bioresour Technol. 2008; 99: 195–199. doi: 10.1016/j.biortech.2006.12.010 17321739

44. Walter V, Syldatk C, Hausmann R. Screening concepts for the isolation of biosurfactant producing microorganisms. In Biosurfactants. Advances in Experimental Medicine and Biology, Sen R. Springer, New York, NY, 2010; 672, pp.1–13. doi: 10.1007/978-1-4419-5979-9_1 20545270

45. Dhiman R, Meena KR, Sharma A, Kanwar SS. Biosurfactants and their screening methods. Res J Recent Sci. 2016; 5: 39–43.

46. Cooper DG, Macdonald CR, Duff SJB, Kosaric N. Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl Environ Microb. 1981; 42: 408–412.

47. Hsieh FC, Li MC, Lin TC. Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR. Curr Microbiol. 2004; 49: 186–191. doi: 10.1007/s00284-004-4314-7 15386102

48. Ismail W, Al-Rowaihi IS, Al-Humam AA, Hamza RY, El Nayal AM, Bououdina M. Characterization of a lipopeptide biosurfactant produced by a crude-oil-emulsifying Bacillus sp. I-15. Int. Biodeter Biodegr. 2013; 84: 168–178.

49. de Franҫa IWL, Lima AP, Lemos JAM, Lemos CGF, Melo VMM, De Santana HB. Production of a biosurfactant by Bacillus subtilis ICA56 aiming bioremediation of impacted soils. Catal Today 2015; 255: 10–15.

50. Cooper DG. Biosurfactants. Mikrobiol. Sci. 1986; 3: 145–149.

51. de Faria AF, Teodoro-Martinez DS, de Oliveira Barbosa GN, Vaz BG, Silva ÍS, Garcia JS, et al. Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry. Process Biochem. 2011; 46: 1951–1957.

52. Bezza FA, Chirwa EMN. Production and applications of lipopeptide biosurfactant for bioremediation and oil recovery by Bacillus subtilis CN2. Biochem Eng J. 2015; 101: 168–178.

53. Al-Wahaibi Y, Joshi S, Al-Bahry S, Elshafie A, Al-Bermani A, Shibula B. Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery. Colloid BioSurf B Biointerfaces 2014; 114: 324–333.

54. Jha SS, Joshi SJ, Geetha SJ. Lipopeptide production by Bacillus subtilis R1 and its possible applications. Braz J Microb 2016; 47: 955–964.

55. Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM, McInerney MJ. Comparison of methods to detect biosurfactant production by diverse microorganisms. J Microbiol Methods 2004; 56: 339–347. doi: 10.1016/j.mimet.2003.11.001 14967225

56. Mulligan CN, Cooper DG, Neufeld RJ. Selection of microbes producing biosurfactants in media without hydrocarbons. J Ferment Technol. 1984; 62: 311–314.

57. Oliveira DWF, Sousa JR, França IWL, Felix AKN, Martins JJL, Gonçalves LRB. Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice. Colloids Surf. B Biointerfaces 2013; 101: 34–43. doi: 10.1016/j.colsurfb.2012.06.011 22796769

58. Amani H, Haghighi M, Keshtkar MJ. Production and optimization of microbial surfactin by Bacillus subtilis for ex situ enhanced oil recovery. Petrol Sci Technol. 2013; 31: 1249–1258.

59. Jokari S, Rashedi H, Amoabediny Gh, Naghizadeh Dilmaghani S, Mazaheri Assadi M. Optimization of surfactin production by Bacillus subtilis ATCC 6633 in a miniaturized bioreactor. Int J Environ Res. 2013; 7: 851–858.

60. Wei Y-H, Wang L-F, Changy J-S, Kung S-S. Identification of induced acidification in iron-enriched cultures of Bacillus subtilis during biosurfactant fermentation. J Biosci Bioeng. 2003; 96: 174–178. doi: 10.1016/s1389-1723(03)90121-6 16233504

61. Haddad NIA, Liu X, Yang S, Mu B. Surfactin isoforms from Bacillus subtilis HSO121: separation and characterization. Protein Peptide Lett. 2008; 15: 265–269.

62. Zhao Y, Yang SZ, Mu BZ. Quantitative analyses of the isoforms of surfactin produced by Bacillus subtilis HSO 121 using GC-MS. Anal Sci. 2012; 28: 789–793. doi: 10.2116/analsci.28.789 22878634

63. Nayarisseri A, Singh P, Singh SK. Screening, isolation and characterization of biosurfactant-producing Bacillus tequilensis strain ANSKLAB04 from brackish river water. Int J Environ Sci Technol. 2018; 1–10.

64. Wang Q, Yu H, Wang M, Yang H, Shen Z. Enhanced biosynthesis and characterization of surfactin isoforms with engineered Bacillus subtilis through promoter replacement and Vitreoscilla hemoglobin co-expression. Process Biochem. 2018; 70: 36–44.

65. Liu Q, Lin J, Wang W, Huang H, Li S. Production of surfactin isoforms by Bacillus subtilis BS-37 and its applicability to enhanced oil recovery under laboratory conditions. Biochem Eng J. 2015; 93: 31–37.

66. Razafindralambo H, Popineau Y, Deleu M, Hbid C, Jacques P, Thonart P, et al. Foaming properties of lipopeptides produced by Bacillus subtilis.  Effect of lipid and peptide structural attributes. Agric Food Chem. 1998; 46: 911–916.

67. Liu JF, Yang J, Yang SZ, Ye RQ, Mu BZ. Effects of different amino acids in culture media on surfactin variants produced by Bacillus subtilis TD7. Appl Biochem Biotechnol. 2012; 166: 2091–2100. doi: 10.1007/s12010-012-9636-5 22415784

68. Nicolas JP. Molecular Dynamics simulation of surfactin molecules at the water-hexane interface. Biophys J. 2003; 85: 1377–1391. doi: 10.1016/S0006-3495(03)74571-8 12944256

69. Ilawe NV, Schweitzer-Stenner R, DiGuiseppi D, Wong M. Is a cross-β-sheet structure of low molecular weight peptides necessary for the formation of fibrils and peptide hydrogel? Phys. Chem. Chem. Phys. 2018; 20: 18158–18168. doi: 10.1039/c8cp00691a 29696249

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