Solubilization of n-hexadecane by micellar solutions of trehalolipid - surfactants of biological origin

Objectives. To isolate biosurfactants of glycolipid nature produced by oil hydrocarbon degrading bacteria and to establish their ability to solubilize hydrophobic compounds in the case of n-hexadecane.
Methods. Trehalolipids were isolated from bacteria Rhodococcus erythropolis X5 (VKM Ac-2532 D) and Rhodococcus erythropolis S67 (VKM Ac-2533 D) included in the MikroBak biopreparation for the bioremediation of oil-contaminated territories. The genome of R. erythropolis X5 is deposited in the National Center for Biotechnology Information database under GenBank accession numbers CP044283 and CP044284, BioSample – SAMN12818508, BioProject – PRJNA573614, and SRA – PRJNA573614. The content of trehalolipid biosurfactants was estimated by the amount of trehalose in aqueous solutions of biosurfactants using the phenolsulfur method. The surface tension of the obtained aqueous solutions of biosurfactants was determined by the du Noüy ring method using a Kruss K6 tensiometer (Kruss, Germany). The critical concentration of micelle formation was determined by the inflection point on the curves of surface tension dependence on the concentration of the biosurfactant solution. In order to establish the solubilizing ability of biosurfactants, the residual concentration of n-hexadecane in an aqueous sample of different concentrations was determined using a gas chromatographic method of analysis.
Results. At a constant surface tension of 24.2 mN/m and 25.0 mN/m for R. erythropolis X5 and R. erythropolis S67, respectively, the critical micelle concentration for both strains was 33 mg/L (3.8 ∙ 10⁻⁵ mol/L). The solubilizing effect of Rhodococcus trehalolipid micellar solutions against hydrophobic n-hexadecane was demonstrated by gas chromatographic analysis. The solubilization process was characterized using molar solubilization capacity (S_m), molar solubilization ratio (MSR), micelle–water partition coefficient (K_m), and solubilization energy 0 (ΔGS ). It was shown that the solubilization process of n-hexadecane proceeds spontaneously 0 (ΔGS = −35.5 kJ/mol) and more efficiently (S_m = 4.3 mol/mol, MSR = 4.7 mol/mol) than in comparison with other biosurfactants of glycolipid nature.
Conclusions. Based on the value of the molar solubilization coefficient, it can be concluded that trehalolipids of the R. erythropolis X5 strain solubilize n-hexadecane in aqueous solutions to a greater extent than compared to other biosurfactants of a glycolipid nature, but are inferior to synthetic surfactants.

1. Alizadeh-Sani M., Hamishehkar H., Khezerlou A., Azizi-Lalabadi M., Azadi Y., Nattagh-Eshtivani E. Bioemulsifiers Derived from Microorganisms: Applications in the Drug and Food Industry. Adv. Pharm. Bull. 2018;8(2):191–199. https://doi.org/10.15171/apb.2018.023

2. Adu S.A., Naughton P.J., Marchant R., Banat I.M. Microbial Biosurfactants in Cosmetic and Personal Skincare Pharmaceutical Formulations. Pharmaceutics. 2020;12(11):1099. https://doi.org/10.3390/pharmaceutics12111099

3. Fenibo E.O., Ijoma G.N., Selvarajan R., Chikere C.B. Microbial Surfactants: The Next Generation Multifunctional Biomolecules for Applications in the Petroleum Industry and Its Associated Environmental Remediation. Microorganisms. 2019;7(11):581. https://doi.org/10.3390/microorganisms7110581

4. Pasternak G., Askitosari T.D., Rosenbaum M.A. Biosurfactants and Synthetic Surfactants in Bioelectrochemical Systems: A Mini-Review. Front. Microbiol. 2020;11:358. https://doi.org/10.3389/fmicb.2020.00358

5. Kumar A., Singh S.K., Kant C., Verma H., Kumar D., Singh P.P., et al. Microbial Biosurfactant: A New Frontier for Sustainable Agriculture and Pharmaceutical Industries. Antioxidants. 2021;10(9):1472. https://doi.org/10.3390/antiox10091472

6. Gouthami K., Mallikarjunaswamy A.M.M., Bhargava R.N., Ferreira L.F.R., Rahdar A., Saratale G.D., Bankole P.O., Mulla S.I. Microbial Biodegradation and Biotransformation of Petroleum Hydrocarbons: Progress, Prospects, and Challenges. In: Genomics Approach to Bioremediation. Genomics Approach to Bioremediation: Principles, Tools, and Emerging Technologies. 2023. P. 229–247. https://doi.org/10.1002/9781119852131.ch13

7. Eras-Muñoz E., Farré A., Sánchez A., Font X., Gea T. Microbial biosurfactants: a review of recent environmental applications. Bioengineered. 2022;13(5):12365–12391. https://doi.org/10.1080/21655979.2022.2074621

8. Claus S., Jenkins Sánchez L., Van Bogaert I.N.A. The role of transport proteins in the production of microbial glycolipid biosurfactants. Appl. Microbiol. Biotechnol. 2021;105(5): 1779–1793. https://doi.org/10.1007/s00253-021-11156-7

9. Sobrinho H.B.S., Luna J.M., Rufino R.D., Porto A.L.F., Sarubbo L.A. Biosurfactants: Classification, properties and environmental applications. In: Recent Developments in Biotechnology. 1st ed. Houston, USA: Studium Press LLC; 2013. P. 303–330.

10. Filonov A.E., Kosheleva I.A., Samoilenko V.A., Shkidchenko A.N., et al. Biological preparation for cleaning of soils from contaminations with oil and oil products, method of its production and application: RU Pat. 2378060. Publ. 10.01.2010 (in Russ.).

11. Delegan Y., Valentovich L., Petrikov K., Vetrova A., Akhremchuk A., Akimov V. Complete Genome Sequence of Rhodococcus erythropolis X5, a Psychrotrophic Hydrocarbon-Degrading Biosurfactant-Producing Bacterium. Microbiol. Resour. Announc. 2019;8(48). https://doi.org/10.1128/mra.01234-19

12. Karimova V.T., Dmitrieva E.D., Nechaeva I.A. The effect of humic substances from different origin peats of the Tula region on the growth of microbial degraders of oil Rhodococcus erythropolis S67 and Rhodococcus erythropolis X5. Izvestiya Tul’skogo gosudarstvennogo universiteta. Estestvennye nauki = News of the Tula State University. Natural Sciences. 2017;2:60–68 (in Russ.).

13. Luong T.M., Ponamoreva O.N., Nechaeva I.A., Petrikov K.V., Delegan Ya.A., Surin A.K., Linklater D., Filonov A.E. Characterization of biosurfactants produced by the oildegrading bacterium Rhodococcus erythropolis S67 at low temperature. World J. Microbiol. Biotechnol. 2018;34(2):20. https://doi.org/10.1007/s11274-017-2401-8

14. Lyong T.M., Nechaeva I.A., Petrikov K.V., Puntus I.F., Ponamoreva O.N. Oil-degrading microorganisms of genus Rhodococcus – potential producers of biosurfactants. Izvestiya vuzov. Prikladnaya khimiya i biotekhnologiya = Proceedings of Universities. Applied Chemistry and Biotechnology. 2016;6(1–16):50–60 (in Russ.).

15. Leonova T.I., Akatova E.V., Puntus I.F. Isolation of glycolipid biosurfactants produced by bacteria Rhodococcus sp. 3-2 by extraction method. Izvestiya Tul’skogo gosudarstvennogo universiteta. Estestvennye nauki = News of the Tula State University. Natural Sciences. 2021;(2):33–41 (in Russ.). https://doi.org/10.24412/2071-6176-2021-2-33-41

16. Yang X., Tan F., Zhong H., Liu G., Ahmad Z., Liang Q. Sub-CMC solubilization of n-alkanes by rhamnolipid biosurfactant: the Influence of rhamnolipid molecular structure. Colloids Surf. B: Biointerfaces. 2020;192:111049. https://doi.org/10.1016/j.colsurfb.2020.111049

17. Lyong T.M., Nechaeva I.A., Petrikov K.V., Filonov A.E., Ponamoreva O.N. Structure and physicochemical properties of glycolipid biosurfactants, produced by oil-degrading bacteria Rhodococcus sp. X5. Izvestiya vuzov. Prikladnaya khimiya i biotekhnologiya = Proceedings of Universities. Applied Chemistry and Biotechnology. 2017;7(2–21):72–79 (in Russ.). https://doi.org/10.21285/2227-2925-2017-7-2-72-79

18. Kuyukina M.S., Ivshina I.B., Philp J.C., Christofi N., Dunbar S.A., Ritchkova M.I. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction. J. Microbiol. Methods. 2001;46(2):149–156. https://doi.org/10.1016/S0167-7012(01)00259-7

19. Ratnikava M.S., Charniauskaya M.I., Bukliarevich H.A., Myamin U.Y., Meloni F., Lussu R., Titok M.A. Rhodococcus erythropolis strain A29-K1 – an effective producer of surface active compounds. In: Biotechnology of Microorganisms: Proc. International Scientific-Practical Conference. 2019. P. 163–165.

20. Petrikov K., Delegan Y., Surin A., Ponamoreva O., Puntus I., Filonov A., Boronin A. Glycolipids of Pseudomonas and Rhodococcus oil-degrading bacteria used in bioremediation preparations: formation and structure. Process Biochem. 2013; 48(5–6):931–935. https://doi.org/10.1016/j.procbio.2013.04.008

21. White D.A., Hird L.C., Ali S.T. Production and characterization of a trehalolipid biosurfactant produced by the novel marine bacterium Rhodococcus sp., strain PML026. J. Appl. Microbiol. 2013;115(3):744–755. https://doi.org/10.1111/jam.12287

22. Yin H., Qiang J., Jia Y., Ye J., Peng H., Qin H., Zhang N., He B. Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater. Process Biochem. 2009;44(3):302–308. https://doi.org/10.1016/j.procbio.2008.11.003

23. Balan S.S., Mani P., Kumar C.G., Jayalakshmi S. Structural characterization and biological evaluation of Staphylosan (dimannooleate), a new glycolipid surfactant produced by a marine Staphylococcus saprophyticus SBPS-15. Enzyme Microb. Technol. 2019;120:1–7. https://doi.org/10.1016/j.enzmictec.2018.09.008

24. Tian Z.J., Chen L.Y., Li D.H., Pang H.Y., Wu S., Liu J.B., Huang L. Characterization of a Biosurfactant-producing Strain Rhodococcus sp. HL-6. Romanian Biotechnol. Lett. 2016;21(4):11650–11659.

25. Philp J.C.M.S., Kuyukina M., Ivshina I., Dunbar S., Christofi N., Lang S., Wray V. Alkanotrophic Rhodococcus ruber as a biosurfactant producer. Appl. Microbiol. Biotechnol. 2002;59(2):318–324. https://doi.org/10.1007/s00253-002-1018-4

26. Tuleva B., Cohen R., Stoev G., Stoineva I. Production and structural elucidation of trehalose tetraesters (biosurfactants) from a novel alkanothrophic Rhodococcus wratislaviensis strain. J. Appl. Microbiol. 2008;104(6):1703–1710. https://doi.org/10.1111/j.1365-2672.2007.03680.x

27. Wang Y., Nie M., Diwu Z., Lei Y., Li H., Bai X. Characterization of trehalose lipids produced by a unique environmental isolate bacterium Rhodococcus qingshengii strain FF. J. Appl. Microbiol. 2019;127(5):1442–1453. https://doi.org/10.1111/jam.14390

28. Dremuk A.P., Kienskaya K.I., Avramenko G.V., Nazarov V.V., Belova I.A. Features of the solubilization action of the binary and ternary surfactant mixtures based on alkyl glucoside. Prikladnaya khimiya i biotekhnologiya = Proceedings of Universities. Applied Chemistry and Biotechnology. 2017;7(1):49–55 (in Russ.). https://doi.org/10.21285/2227-2925-2017-7-1-50-56

29. Zarueva E.S., Nechaeva I.A., Ponamoreva O.N. Solubilization of n-hexadecane in a mineral medium with surfactants. Izvestiya Tul’skogo gosudarstvennogo universiteta. Estestvennye nauki = News of the Tula State University. Natural Sciences. 2020;(1):3–12 (in Russ.).

30. Harendra S., Vipulanandan C. Effects of surfactants on solubilization of perchloroethylene (PCE) andtrichloroethylene (TCE). Ind. Eng. Chem. Res. 2011;50(9):5831–5837. https://doi.org/10.1021/ie102589e

31. Rodrigues R., Betelu S., Colombano S., Masselot G., Tzedakis T., Ignatiadis I. Influence of temperature and surfactants on the solubilization of hexachlorobutadiene and hexachloroethane. J. Chem. Eng. Data. 2017;62(10): 3252–3260. https://doi.org/10.1021/acs.jced.7b00320

32. Li S., Pi Y., Bao M., Zhang C., Zhao D., Li Y., Sun P., Lu J. Effect of rhamnolipid biosurfactant on solubilization of polycyclic aromatic hydrocarbons. Marine Pollut. Bull. 2015;101(1):219–225. https://doi.org/10.1016/j.marpolbul.2015.09.059

33. Zhong H., Liu Y., Liu Z., Jiang Y., Tan F., Zeng G., Yuan X., Yan M., Niu Q., Liang Y. Degradation of pseudo-solubilized and mass hexadecane by a Pseudomonas aeruginosa with treatment of rhamnolipid biosurfactant. Int. Biodeterior. Biodegradation. 2014;94:152–159. https://doi.org/10.1016/j.ibiod.2014.07.012

34. Yang Z., Cui J., Yin B. Solubilization of Nitrogen Heterocyclic Compounds Using Different Surfactants. Water, Air, Soil Pollut. 2018;229(9):304. https://doi.org/10.1007/s11270-018-3917-8