Investigation on spectral and biomedical characterization of rhamnolipid from a marine associated bacterium Pseudomonas aeruginosa (DKB1)

Abstract

Bio-surfactants are a principal group of significant molecules obtained from the microbial sources expressed with distinctive characteristics like biodegradation of hydrocarbons and also have different biomedical properties. The present investigation aims to assess the biomedical properties of synthesized bio-surfactant, rhamnolipid from Pseudomonas aeruginosa (DKB1) under in vitro conditions. The candidate bacterium P. aeruginosa (DKB1) was isolated from oil-polluted fishing harbors of Kanyakumari coast. Initially, the bio-surfactant production by this candidate strain was confirmed by oil displacement assay, hemolytic assay, drop collapse assay and emulsification index. Further, the production of bio-surfactant was achieved through submerged fermentation process using Bushnell–Haas mineral salts medium supplemented with 2% olive oil. The yield of the bio-surfactant was attained as 2.4 g/l and confirmed as rhamnolipid through blue agar plate assay; further, the extracted rhamnolipid was purified and characterized through standard procedures. In stability studies, the rhamnolipid could withstand up to pH 12, temperature 100 °C and 15% of NaCl concentration. The biomedical application of rhamnolipid (30 μg ml−1) was determined by antibacterial, antioxidant and cytotoxic studies. It exhibited a maximum growth inhibition against Bacillus subtilis (26 mm) with the MIC value of 8 μg ml−1. In antioxidant test, rhamnolipid expressed significant (P < 0.0001) inhibition of total reducing power (44.11%), DPPH activity (61.60%), hydroxyl radical (83.30%) and nitric oxide (51.86%) scavenging ability at 100 μg ml−1with the respective IC50 values of 130.50, 77.18, 52.08 and 95.43 μg ml−1. The anticancer activity of the rhamnolipid was assessed with the help of MTT test against MCF-7, HT-29 and E-143 cancer cell lines individually, and the viability of the cells was observed, respectively, as 10.24, 17.66 and 13.50% at 250 μg ml−1concentration with the respective IC50 values of 140.2, 81.02 and 138.9 μg ml−1. From the results, it could be concluded that the rhamnolipid produced by P. aeruginosa (DKB1) isolated from oil-polluted area has effective biomedical properties.

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References

  1. Abeer Mohammed AB, Tayel AA, Elguindy NM (2018) Production of new rhamnolipids Rha C16–C16 by Burkholderia sp. through biodegradation of diesel and biodiesel. Beni Suef Univ J Basic Appl Sci 7:492–498

    Google Scholar 

  2. Abouseoud M, Maachi R, Amrane A, Boudergua S, Nabi A (2008) Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens. Desalination 223:143–151

    CAS  Article  Google Scholar 

  3. Ahmad Z, Zhang X, Imran M, Zhong H, Andleeb S, Zulekha R, Ahmad I, Coulon F (2021) Production, functional stability, and effect of rhamnolipid biosurfactant from Klebsiella sp. on phenanthrene degradation in various medium systems. Ecotoxicol Environ Saf 207:1–12

    Article  CAS  Google Scholar 

  4. Akinjogunla OJ, Eghafona NO, Enabulele IO, Mboto CI, Ogbemudia FO (2010) Antibacterial activity of ethanolic extracts of Phyllanthus amarus against extended spectrum β-lactamase producing E. coli isolated from stool samples of HIV sero-positive patients with or without diarrhea. Afr J Pharm Pharmacol 4:402–407

    Google Scholar 

  5. Anandarj B, Thivakaran P (2010) Isolation and production of biosurfactant producing organism from oil spilled soil. J Biosci Tech 1:120–126

    Google Scholar 

  6. Balan SS, Ganesh Kumarb C, Jayalakshmi S (2017) Aneurinifactin, a new lipopeptide biosurfactant produced by a marine Aneurinibacillus aneurinilyticus SBP-11 isolated from Gulf of Mannar: purification, characterization and its biological evaluation. Microbiol Res 194:1–9

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53:495–508

    CAS  PubMed  Article  Google Scholar 

  8. Bharali P, Konwar BK (2011) Production and physico-chemical characterization of a biosurfactant produced by Pseudomonas aeruginosa OBP1 isolated from petroleum sludge. Appl Biochem Biotechnol 164:1444–1460

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. Bhat R, Dayamani KJ, Hathwar S, Hegde R, Kush A (2015) Exploration on production of rhamnolipid biosurfactants using native Pseudomonas aeruginosa strains. J BioSci Biotechnol 4:157–166

    Google Scholar 

  10. Chang ST, Wu JH, Wang SY, Kang PL, Yang NS, Shyur LF (2001) Antioxidant activity of extracts from Acacia confusa bark and heartwood. J Agric Food Chem 49:3420–3424

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. Cooper DG, Goldenberg BG (1987) Surface-active agents from two Bacillus sp. Appl Environ Microbiol 53:224–229

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Darvishi F, Destain J, Nahvi I, Thonart P, Zarkesh-Esfahani H (2011) High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate. New Biotechnol 28:756–760

    CAS  Article  Google Scholar 

  13. Deivakumari M, Sanjivkumar M, Suganya AM, Ruban Prabakaran J, Palavesam A, Immanuel G (2020) Studies on reclamation of crude oil polluted soil by biosurfactant producing Pseudomonas aeruginosa(DKB1). Biocatal Agric Biotechnol 29:1–12

    Article  Google Scholar 

  14. Ferrera-Rodriguez O, Greer CW, Juck D, Consaul LL, Martinez- Romero E (2012) Hydrocarbon-degrading potential of microbial communities from Arctic plants. J Appl Microbiol 114:71–83

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  15. Folch JM, Lees M, Stanly HS (1956) A simple method for the isolation and quantification of total lipids from animal tissues. J Biol Chem 226:497–509

    Article  Google Scholar 

  16. Garg M, Priyanka S, Chatterjee M (2018) Isolation, characterization and antibacterial effect of biosurfactant from Candida parapsilosis. Biotechnol Rep 18:1–6

    Google Scholar 

  17. Gogoi D, Bhagowati P, Gogoi P, Bordoloi N, Rafay A, Doloui S, Mukherjee AK (2016) Structural and physico-chemical characterization of a dirhamnolipid biosurfactant purified from Pseudomonas aeruginosa: application of crude biosurfactant in enhanced oil recovery. RSC Adv 74:11–36

    Google Scholar 

  18. Gomaa EZ (2012) Antimicrobial activity of a biosurfactant produced by Bacillus licheniformis strain M104 grown on whey. Afr J Microbiol Res 6:4396–4403

    CAS  Google Scholar 

  19. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite and (15N) nitrate in biological fluids. Anal Biochem 126:131–138

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. Halliwell B, Gutteridge JMC, Aruoma OI (1987) The deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165:215–219

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. Hoskova M, Schreiberova O, Jezdik R, Chudoba J, Masak J, Sigler K, Rezanka T (2013) Characterization of rhamnolipids produced by nonpathogenic Acinetobacter and Enterobacter bacteria. Bioresour Technol 130:510–516

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. Ibrahim HMM (2016) Biodegradation of used engine oil by novel strains of Ochrobactrum anthropi HM-1 and Citrobacter freundii HM-2 isolated from oil contaminated soil. Egypt J Pet 2:1–13

    Google Scholar 

  23. Jadhav V, Yadav A, Shouche Y, Aphale S (2013) Studies on biosurfactant from Oceanobacillus sp. BRI 10 isolated from Antarctic sea water. Desalination 318:64–71

    CAS  Article  Google Scholar 

  24. Janek T, Lukaszewicz M, Rezanka T, Krasowska A (2010) Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescensBD5 isolated from water from the Arctic Archipelago of Svalbard. Bioresour Technol 101:6118–6123

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. Jemil N, Ben Ayed H, Manresa A, Nasri M, Hmidet N (2017) Antioxidant properties, antimicrobial and anti-adhesive activities of DCS1 lipopeptides from Bacillus methylotrophicus DCS1. BMC Microbiol 17:144–155

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Jenkin TC, Thies EJ, Mosley EE (2001) Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples. J Agric Food Chem 49:2142–2145

    Article  CAS  Google Scholar 

  27. Joshi PA, Shekhawat DB (2014) Screening and isolation of biosurfactant producing bacteria from petroleum contaminated soil. Eur J Exp Biol 4:164–169

    Google Scholar 

  28. Karkera K, Pendse A, Aruna K (2012) Studies of Biosurfactant production by Pseudomonas aeruginosa R2 isolated from oil contaminated soil sample. Asian J Exp Biol Sci 7:123–129

    Google Scholar 

  29. Khopade A, Ren B, Liu X, Mahadik K, Zhang L, Kokare C (2012) Production and characterization of biosurfactant from marine Streptomyces sp.B3. J Colloid Interface Sci 367:311–318

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. Kim HS, Yoon BD, Lee CH, Suh HH, Oh HM, Katsuragi T, Tani Y (1997) Production and properties of a lipopeptide biosurfactant from Bacillus subtilis C9. J Ferment Bioeng 84:41–46

    Article  Google Scholar 

  31. Lan G, Fan Y, Liu C, Chen C, Li G, Liu Y, Yu X (2015) Rhamnolipid production from waste cooking oil using Pseudomonas SWP-4. Biochem Eng J 101:44–54

    CAS  Article  Google Scholar 

  32. Mani P, Dineshkumar G, Jayaseelan T, Deepalakshmi K, Ganesh Kumar C, Senthil Balan S (2016) Antimicrobial activities of a promising glycolipid biosurfactant from a novel marine Staphylococcus saprophyticus SBPS 15. 3 Biotech 6:163

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Merghni A, Dallel I, Noumi E, Kadmi Y, Hentati H, Tobji S, Amor A, Mastouri M (2017) Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureus strains. Microb Pathog 104:84–89

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. Mijin DZ, Milic BD, Misic-Vukovic MM (2006) Synthesis of substituted 3-cyano-2-pyridones: part IV. Influence of 3-alkyl-2,4-pentadione and N-alkyl cyanoacetamide structure on the enzyme catalyzed synthesis of substituted 3-cyano2-pyridones. Indian J Chem 45B:993–1003

    CAS  Google Scholar 

  35. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. Muller MM, Hausmann R (2011) Regulatory and metabolic network of rhamnolipid biosynthesis: traditional and advanced engineering towards biotechnological production. Appl Microbiol Biotechnol 91:251–264

    PubMed  Article  CAS  Google Scholar 

  37. Nieva-Echevarria B, Manzanos MJ, Goicoechea E, Guillen MD (2015) 2,6-Di-tert-butylhydroxytoluene and its metabolites in foods. Comp Rev Food Sci Food Saf 14:67–80

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. Oyaizu M (1986) Antioxidative activity of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography. Nippon Shokuhin Kogyo Gakkaishi 35:771–775

    Article  Google Scholar 

  39. Parisa E, Hamidreza H, Shayesteh B (2020) Recent advancements in the production of rhamnolipid biosurfactants by Pseudomonas aeruginosa. RSC Adv 10:34014–34032

    Article  Google Scholar 

  40. Patel RM, Desai AJ (1997) Biosurfactant production by Pseudomonas aeruginosa GS3 from molasses. Lett Appl Microbiol 25:91–94

    CAS  Article  Google Scholar 

  41. Pereira JFB, Gudina EJ, Doria ML, Domingues MR, Rodrigues LR, Teixeira JA (2012) Characterization by electrospray ionization and tandem mass spectrometry of rhamnolipids produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oil. Eur J Mass Spectrom 18:399–406

    CAS  Article  Google Scholar 

  42. Pornsunthorntawee O, Wongpanit P, Chavadej S, Abe M, Rujiravanit R (2008) Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum contaminated soil. Bioresour Technol 99:1589–1595

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. Priya T, Usharani G (2009) Comparative study for biosurfactant production by using Bacillus subtilis and Pseudomonas aeruginosa. Bot Res Int 2:284–287

    CAS  Google Scholar 

  44. Relman DA (1993) Universal bacterial 16S rDNA amplification and sequencing. In Diagnostic molecular microbiology, Edited by Persing DH, Smith TF, Tenover FC, White TJ. Washington, DC. pp. 489–495.

  45. Rodrigues LR, Moldes A, Teixeira J (2006) Kinetic study of fermentative biosurfactant production by Lactobacillus strains. Biochem Eng J 28:109–116

    CAS  Article  Google Scholar 

  46. Saikia RR, Deka S, Deka M, Banat IM (2012) Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production. Ann Microbiol 62:753–763

    CAS  Article  Google Scholar 

  47. Saravanan V, Vijayakumar S (2012) Isolation and screening of biosurfactant producing microorganisms from oil contaminated soil. J Acad Indus Res 1:5

    Google Scholar 

  48. Satpute SK, Bhawasar BD, Dhakephalkar PK, Chopade BA (2008) Assessment of different screening method for selecting biosurfactant producing marine bacteria. Indian J Geo Mar Sci 37:243–250

    CAS  Google Scholar 

  49. Senthil Balan S, Jayalakshmi S (2013) Glycolipid biosurfactant production using low cost medium from marine bacterium Pseudomonas aeruginosa of Mudasalodai coast. Int J Green Chem Bioprocess 3:33–37

    Google Scholar 

  50. Sharma D, Saharan BS, Chauhan N, Bansal A, Procha S (2014) Production and structural characterization of Lactobacillus helveticus derived biosurfactant. Sci World J 1:1–9

    Google Scholar 

  51. Sharma S, Verma R, Pandey LM (2019) Crude oil degradation and biosurfactant production abilities of isolated Agrobacterium fabrum SLAJ731. Biocatal Agric Biotechnol 21:1–10

    CAS  Google Scholar 

  52. Shoeb E, Ahmed N, Akhter J, Badar U (2015) Screening and characterization of biosurfactant-producing bacteria isolated from the Arabian Sea coast of Karachi. Turk J Biol 39:210–216

    CAS  Article  Google Scholar 

  53. Siegmund I, Wagner F (1991) New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5:265–268

    CAS  Article  Google Scholar 

  54. Sivapathasekaran C, Das P, Mukherjee S, Saravanakumar J, Mandal M, Sen R (2010) Marine bacterium derived lipopeptides: characterization and cytotoxic activity against cancer cell lines. Int J Pept Res Ther 16:215–222

    CAS  Article  Google Scholar 

  55. Stanghellini ME, Miller RM (1997) Biosurfactants: their identity and potential efficacy in the biological control of zoosporic plant pathogens. Plant Dis 81:4–12

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. Tabbene O, Gharbi D, Slimene IB, Elkahoui S, Alfeddy MN, Cosette PML, Mangoni T, Jouenne LF (2012) Antioxidative and DNA protective effects of bacillomycin D-like lipopeptides produced by B38 strain. Appl Biochem Biotechnol 168:2245–2256

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  57. Tahzibi A, Kamal F, Assadi MM (2004) Improved production of rhamnolipids by a Pseudomonas aeruginosa mutant. Iran Biomed J 8:25–31

    CAS  Google Scholar 

  58. Takahashi M, Mortia T, Fukuoka T, Imura T, Kitamoto D (2012) Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effect against H2O2 induced oxidative stress in cultured human skin fibroblasts. J Oleo Sci 61:457–464

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. Tamura K, Dudley J, Nei M, Kumar SM (2007) Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1605

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  60. Thavasi R, Jayalakshmi S, Balasubramanian T, Banat IM (2008) Production and characterization of a glycolipid biosurfactant from Bacillus megaterium using economically cheaper sources. World J Microbiol Biotechnol 24:917–925

    CAS  Article  Google Scholar 

  61. Velmurugan M, Baskaran A, Dinesh Kumar S, Sureka I, Arockia Raj E, Emelda J, Sathiyamurthy K (2015) Screening, stability and antibacterial potential of rhamnolipids from Pseudomonas sp., isolated from hydrocarbon contaminated soil. J Appl Pharm 5:026–033

    CAS  Google Scholar 

  62. Vijayakumar S, Saravanan V (2015) Biosurfactants-types, sources and applications. Res J Microbiol 10:181–192

    Article  Google Scholar 

  63. Waghmode S, Swami S, Sarkar D, Suryavanshi M, Roachlani S, Choudhari P, Satpute S (2020) Exploring the pharmacological potentials of biosurfactant derived from Planococcus maritimus SAMP MCC 3013. Curr Microbiol 77:452–459

    CAS  PubMed  Article  Google Scholar 

  64. Yalcın E, Cavusoglu K (2010) Structural analysis and antioxidant activity of a biosurfactant obtained from Bacillus subtilis RW-I. Turk Biyokim Derg 35:243–247

    Google Scholar 

  65. Yuliania H, Perdani MS, Savitri I, Manurung M, Sahlan M, Wijanarko A, Hermansyah H (2018) Antimicrobial activity of biosurfactant derived from Bacillus subtilis C19. Energy Procedia 153:274–278

    Article  CAS  Google Scholar 

  66. Zouari R, Moalla-Rekik D, Sahnoun Z, Rebai T, Ellouze-Chaabouni S, Ghribi-Ayid D (2016) Evaluation of dermal wound healing and in vitro antioxidant efficiency of Bacillus subtilis SPB1 biosurfactant. Biomed Pharmacother 84:878–891

    CAS  PubMed  Article  PubMed Central  Google Scholar 

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Acknowledgements

The authors are gratefully acknowledge the DST-SERB, New Delhi, Govt. of India, for its financial support in the form of a Research grant (Grant no. EMR/2017/001453).

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Sanjivkumar, M., Deivakumari, M. & Immanuel, G. Investigation on spectral and biomedical characterization of rhamnolipid from a marine associated bacterium Pseudomonas aeruginosa (DKB1). Arch Microbiol (2021). https://doi.org/10.1007/s00203-021-02220-x

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Keywords

  • Pseudomonas sp.
  • Biosurfactant
  • Rhamnolipid
  • Antioxidant activity
  • Anticancer activity