MICROBIAL PRODUCTION OF BIOSURFACTANTS AND THEIR BIOCHEMICAL CHARACTERIZATION

Authors

  • HARISH THUMMALA Medical Microbiologist, Micro Laboratory, Mutyalareddy Nagar, Amaravathi Road, Guntur, Andhra Pradesh, India
  • PRIYANKA K Department of Obstetrics and Gynaecology, Vinayaka Mission’s Kirupananda Variyar Medical College and Hospitals, Vinayaka Missions Research Foundation (Deemed to be University), Seeragapadi, Sankari Main Road, Salem, Tamil Nadu, India.
  • SURESH BABU SAYANA Department of Pharmacology, Government Medical College, Bhadradri Kothagudem, Telangana, India
  • RAMESH KANDIMALLA Department of Biochemistry, Government Medical College, Narsampet, Warangal, Telangana, India

DOI:

https://doi.org/10.22159/ajpcr.2025v18i11.56591

Keywords:

Biosurfactant, microbial production, emulsification index, surface tension, biochemical characterization, sustainable surfactants

Abstract

Objective: The present work was undertaken to isolate an efficient biosurfactant-producing bacterium, optimize its production under different environmental and nutritional conditions, and characterize the structural and functional properties of the biosurfactant for prospective industrial use.

Methods: Environmental samples from soil and wastewater were subjected to preliminary screening through hemolytic activity, oil displacement, and emulsification index (E24) assays. The most promising isolate was identified by morphological and biochemical tests, followed by molecular confirmation using 16S rRNA gene sequencing. Production parameters were optimized by varying carbon and nitrogen sources, culture pH, temperature, and incubation period. Crude biosurfactant was recovered through acid precipitation and solvent extraction. Its functional attributes were examined through surface tension reduction, critical micelle concentration (CMC) determination, emulsification potential, and stability across pH, temperature, and salinity gradients. Structural features were analyzed by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography– mass spectrometry.

Results: The identified Bacillus subtilis strain effectively reduced surface tension from 72.0 to 28.6 mN/m and exhibited a high emulsification index (75.4±1.2% with kerosene). Optimal production occurred with glucose (2% w/v) and ammonium nitrate (0.5% w/v) at pH 7 and 30°C after 72 h, yielding 4.85±0.15 g/L of crude biosurfactant. The CMC was estimated at 35 mg/L. The compound retained stability between pH 4–10, at temperatures up to 100°C, and in salinity up to 10% NaCl. FTIR and GC–MS confirmed a glycolipid nature, composed mainly of long-chain fatty acids and sugar moieties.

Conclusion: The biosurfactant derived from B. subtilis displayed strong surface-active properties, appreciable yield, and robustness under diverse physicochemical conditions. These features underline its promise for applications in petroleum recovery, bioremediation, effluent treatment, and biopharmaceutical formulations, underscoring its potential as a safe and sustainable alternative to synthetic surfactants.

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References

1. 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, PMID 25566213

2. Marchant R, Banat IM. Microbial biosurfactants: Challenges and opportunities for future exploitation. Trends Biotechnol. 2012;30(11):558-65. doi: 10.1016/j.tibtech.2012.07.003, PMID 22901730

3. Santos DK, Rufino RD, Luna JM, Santos VA, Sarubbo LA. Biosurfactants: Multifunctional biomolecules of the 21st century. Int J Mol Sci. 2016;17(3):401. doi: 10.3390/ijms17030401, PMID 26999123

4. Jimoh AA, Lin J. Biosurfactant: A new frontier for greener technology and environmental sustainability. Ecotoxicol Environ Saf. 2019;184:109607. doi: 10.1016/j.ecoenv.2019.109607, PMID 31505408

5. Mulligan CN. Environmental applications for biosurfactants. Environ Pollut. 2005;133(2):183-98. doi: 10.1016/j.envpol.2004.06.009, PMID 15519450

6. Abdel-Mawgoud AM, Lépine F, Déziel E. Rhamnolipids: Diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol. 2010;86(5):1323-36. doi: 10.1007/s00253-010-2498-2, PMID 20336292

7. Purwasena IA, Amaniyah M, Astuti DI, Firmansyah Y, Sugai Y. Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: A green chemical for microbial enhanced oil recovery (MEOR). Sci Rep. 2024 May 4;14(1):10270. doi: 10.1038/ s41598-024-61096-1, PMID 38704438

8. Singh P, Cameotra SS. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol. 2004;22(3):142-6. doi: 10.1016/j.tibtech.2004.01.010, PMID 15036865

9. Souza EC, Vessoni-Penna TC, De Souza Oliveira RP. Biosurfactant-enhanced hydrocarbon bioremediation: An overview. Int Biodeterior Biodegrad. 2014;89:88-94. doi: 10.1016/j.ibiod.2014.01.007

10. Henkel M, Müller MM, Kügler JH, Lovaglio RB, Contiero J, Syldatk C, et al. Rhamnolipids as biosurfactants from renewable resources: Concepts for next-generation rhamnolipid production. Process Biochem. 2012;47(8):1207-19. doi: 10.1016/j.procbio.2012.04.018

11. Geetha SJ, Banat IM, Joshi SJ. Biosurfactants: Production and potential applications in microbial enhanced oil recovery (MEOR). Biocatal Agric Biotechnol. 2018;14:23-32. doi: 10.1016/j.bcab.2018.01.010

12. Shah MU, Moniruzzaman M, Sivapragasam M, Talukder MM, Yusup S, Goto M. Production and applications of microbial biosurfactants for sustainable agriculture. Sustainability. 2021;13(18):9948. doi: 10.3390/ su13189948

13. Chabhadiya S, Acharya DK, Mangrola A, Shah R, Pithawala EA. Unlocking the potential of biosurfactants: Innovations in metabolic and genetic engineering for sustainable industrial and environmental solutions. Biotechnol Notes. 2024;5:111-9. doi: 10.1016/j. biotno.2024.07.001, PMID 39416688

14. Kubicki S, Bollinger A, Katzke N, Jaeger KE, Loeschcke A, Thies S. Marine biosurfactants: Biosynthesis, structural diversity and biotechnological applications. Mar Drugs. 2019 Jul 9;17(7):408. doi: 10.3390/md17070408, PMID 31323998

15. Ramyabharathi S, Sankari Meena KS, Rajendran L, Karthikeyan G, Jonathan EI, Raguchander T. Biocontrol of wilt-nematode complex infecting gerbera by Bacillus subtilis under protected cultivation. Egypt J Biol Pest Control. 2018;28(1):21. doi: 10.1186/s41938-018-0027-2

16. Bodour AA, Miller-Maier RM. Application of a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol Methods. 1998;32(3):273-80. doi: 10.1016/S0167-7012(98)00031-1, PMID 9631546

17. Carrillo PG, Mardaraz C, Pitta-Alvarez SI, Giulietti AM. Isolation and selection of biosurfactant-producing bacteria. World J Microbiol Biotechnol. 1996;12(1):82-4. doi: 10.1007/BF00327807, PMID 24415095

18. Morikawa M, Daido H, Takao T, Murata S, Shimonishi Y, Imanaka T. A new lipopeptide biosurfactant produced by Bacillus subtilis strain BS-3. J Bacteriol. 1993;175(20):6459-66. doi: 10.1128/jb.175.20.6459- 6466.1993, PMID 8407822

19. 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(3):339-47. doi: 10.1016/j.mimet.2003.11.001, PMID 14967225

20. Cooper DG, Goldenberg BG. Surface-active agents from two Bacillus species. Appl Environ Microbiol. 1987;53(2):224-9. doi: 10.1128/ aem.53.2.224-229.1987, PMID 16347271

21. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697-703. doi:10.1128/jb.173.2.697-703.1991. PMID:1987160

22. Thavasi R, Jayalakshmi S, Banat IM. Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri, and Pseudomonas aeruginosa. Bioresour Technol. 2011;102(2):772-8. doi: 10.1016/j. biortech.2010.08.099, PMID 20863694

23. Dong H, Zheng A, He Y, Wang X, Li Y, Yu G, et al. Optimization and characterization of biosurfactant produced by indigenous Brevibacillus borstelensis isolated from a low permeability reservoir for application in MEOR. RSC Adv. 2022 Jan 12;12(4):2036-47. doi: 10.1039/ d1ra07663a, PMID 35425221

24. Nitschke M, Costa SG. Biosurfactants in food industry. Trends Food Sci Technol. 2007;18(5):252-9. doi: 10.1016/j.tifs.2007.01.002

25. Mnif I, Ghribi D. Review lipopeptides biosurfactants: Mean classes and new insights for industrial, biomedical, and environmental applications. Peptides. 2015;71:100-25. doi: 10.1016/j.peptides.2015.07.006, PMID 26210856

26. Zhang Y, Placek TL, Jahan R, Alexandridis P, Tsianou M. Rhamnolipid Micellization and Adsorption Properties. Int J Mol Sci. 2022; 23(19):11090. doi: 10.3390/ijms231911090.

27. Safari P, Hosseini M, Lashkarbolooki M, Ghorbani M, Najafpour Darzi G. Evaluation of surface activity of rhamnolipid biosurfactants produced from rice bran oil through dynamic surface tension. J Petrol Explor Prod Technol. 2023;13(7):2139-53. doi:10.1007/s13202-023- 01660-z

28. Dini S, Bekhit AE, Roohinejad S, Vale JM, Agyei D. The physicochemical and functional properties of biosurfactants: A review. Molecules. 2024;29(11):2544. doi: 10.3390/molecules29112544, PMID 38893420

29. Albasri HM, Almohammadi AA, Alhhazmi A, Bukhari DA, Waznah MS, Mawad AMM. Production and characterization of rhamnolipid biosurfactant from thermophilic Geobacillus stearothermophilus bacterium isolated from Uhud mountain. Front Microbiol. 2024;15:1358175. doi:10.3389/fmicb.2024.1358175

30. Mishra A, Tiwari P, Pandey LM. Surface, interfacial and thermodynamic aspects of the rhamnolipid-salt systems. J Mol Liq. 2023;384:122245. doi:10.1016/j.molliq.2023.122245

31. Shaikhah D, Loise V, Angelico R, Porto M, Calandra P, Abe AA, et al. New trends in biosurfactants: From renewable origin to green enhanced oil recovery applications. Molecules. 2024;29(2):301. doi: 10.3390/ molecules29020301. PMID: 38257213

32. Thakur V, Verma P, Awasthi A. Recent progress in microbial biosurfactants production strategies. Bioresour Technol. 2024;397:130222.

33. Markam SS, Yadav P, Singh R. Microbial biosurfactants: Green alternatives and opportunities. Sustain Chem Pharm. 2024;37:101433.

34. Qamar SA, Pacifico S. Cleaner production of biosurfactants via bio-waste valorization: A comprehensive review of characteristics, challenges, and opportunities in bio-sector applications. J Environ Chem Eng. 2023;11(12):111555. doi:10.1016/j.jece.2023.111555

35. Hsu CY, Wang JH. Biosurfactants: Properties, applications and emerging trends. J King Saud Univ Sci. 2025;37:102714.

36. Campos JM, Stamford TL, Sarubbo LA, De Luna JM, Rufino RD, Banat IM. Microbial biosurfactants as additives for food industries. Biotechnol Prog. 2013;29(5):1097-108. doi: 10.1002/btpr.1796, PMID 23956227

37. Pruthi V, Cameotra SS. Rapid method for monitoring maximum biosurfactant production and determining media component interaction effects. J Ind Microbiol Biotechnol. 1997;19(4):235-9.

38. Haba E, Espuny MJ, Busquets M, Manresa A. Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils. J Appl Microbiol. 2000;88(3):379-87. doi: 10.1046/j.1365-2672.2000.00961.x, PMID 10747218

Published

07-11-2025

How to Cite

HARISH THUMMALA, et al. “MICROBIAL PRODUCTION OF BIOSURFACTANTS AND THEIR BIOCHEMICAL CHARACTERIZATION”. Asian Journal of Pharmaceutical and Clinical Research, vol. 18, no. 11, Nov. 2025, pp. 201-9, doi:10.22159/ajpcr.2025v18i11.56591.

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