Isolation of Surfactants Synthesized by the Pseudomonas Bacteria and Study of Their Properties




Rhamnolipid surfactants, Physicochemical properties, Acid precipitation, Plant growth stimulators


Background. The important problem of biosurfactants production is biosynthesis optimization. But lack of effective isolation methods elaboration with simultaneous new costeffective products is the greatest weaknesses of existing technologies.

Objective. The aim of the study is rational technology elaboration for isolation of biosurfactants obtained from strains Pseudomonas sp. PS-17 and P. fluorescens 8573. Investigation of the influence of different acids and temperatures on the efficiency of isolation of the surfactant products, study the properties of the obtained products. Determination of the possible directions of the use of supernatant obtained after precipitation of the biocomplexes (SPL) as a new inexpensive product.

Methods. Rhamnolipid surfactant concentrate was precipitated from culture liquid supernatant (CLS) by acidification to pH 3-4 with acid solutions (HCl, H2SO4, H3PO4, HNO3, CH3COOH), kept at 100 °C for 25 min, cooled to room temperature, centrifuged before the phase separation, the supernatant was decanted. The surface tension of the SPL was determined by du Noüy method (with a platinum ring). The emulsification index of biosurfactants was determined regarding mineral oil and sunflower oil. The RLs were isolated from the SPL by extraction with a mixture of ethyl acetate and isopropanol, their composition was determined by thin layer chromatography. Influence on plants was assessed by their morphometric parameters after presowing seed treatment.

Results. The rational technology for surfactants isolation from strains Pseudomonas sp. PS-17 and P. fluorescens 8573 was developed It was shown that the suitable method of the isolation of the biosurfactants of Pseudomonas sp. PS-17 and P. fluorescens 8573 is acidic precipitation from CLS with heating. As a result, the product yield was increased by 20%, and the duration of the process was reduced. The physico-chemical properties of the SPLs after the isolation of biosurfactants from the CLS were studied. SPLs have been shown to be effective oil emulsifiers, foaming and wetting agents for various surfaces. It was shown that SPLs (at dilutions 1:10) do not exhibit phytotoxic effects and stimulate the growth of watercress.

Conclusions. The new wasteless technology for Pseudomonas strains biosurfactants isolation has been proposed, which provides for the elimination of the extraction stage with solvents, as a result, the yield of the target products has been increased. Thus, the technology has economic and environmental advantages. It was shown that SPLs, being inexpensive and effective products, can be used in environmentally friendly technologies: in agriculture (for stimulation of plant growth), for remеdiation of contaminated soils, production of detergent compositions.


Mir Sh, Jamal P, Alama MdZ, Mir AB, Ansari AH. Microbial surface tensio-active compounds: production and industrial application perspectives. Int J Biotechnol Bioeng. 2017;3(8):273-92.

Chong H, Li Q. Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact. 2017;16:13. DOI: 10.1186/s12934-017-0753-2.

Lotfabad T, Ebadipour N, RoostaAzad R. Evaluation of a recycling bioreactor for biosurfactant production by Pseudomonas aeruginosa MR01 using soybean oil waste. J Chem Technol Biotechnol. 2016;91(5):1368-77. DOI: 10.1002/jctb.4733

Gudiña E, Rodrigues A, de Freitas V, Azevedo Z, Teixeira J, Rodrigues L. Valorization of agro-industrial wastes towards the production of rhamnolipids. Biores Technol. 2016;212:144-50. DOI: 10.1016/j.biortech.2016.04.027

Li Q. Rhamnolipid synthesis and production with diverse resources. Front Chem Sci Eng. 2017;11(1):27-36. DOI: 10.1007/s11705-016-1607-x

Yerokhin V, Pokynbroda Т, Karpenko O, Novikov V. Study of the growth and synthesis of the target product by the strain Pseudomonas species PS-17 – producent of extracellular biosurfactants. Visnyk Natsionalnoho Universytetu Lvivska Politekhnika. 2006;553:124-7.

Pokynbroda Т, Karpenko О. Synthesis of biosurfactants by Pseudomonas strains on biodiesel production waste. Proceedings of Internet Conference Biotechnology: Experience, Traditions and Innovations; 2016 December. p. 155-60. Available from:

Kaskatepe B, Yildiz S, Gumustas M, Ozkan S. Biosurfactant production by Pseudomonas aeruginosa in kefir and fish meal. Braz J Microbiol. 2015;46(3):855-9. DOI: 10.1590/S1517-838246320140727

Henkel M, Schmidberger A, Vogelbacher M, Kühnert C, Beuker J, Bernard T, et al. Kinetic modeling of rhamnolipid production by Pseudomonas aeruginosa PAO1 including cell density-dependent regulation. Appl Microbiol Biotechnol. 2014;98(16):7013-25. DOI: 10.1007/s00253-014-5750-3

Karpenko OV, Pokynbroda TYa, Makitra RG. Optimal methods for the isolation of biogenic surface-active rhamnolipids. Zhurnal Obschej Khimii. 2009;79(12):2011-4.

Beuker J, Steier A, Wittgens A, Rosenau F, Henkel M, Hausmann R. Integrated foam fractionation for heterologous rhamnolipid production with recombinant Pseudomonas putida in a bioreactor. Amb Express. 2016;6:11. DOI: 10.1186/s13568-016-0183-2

Scott M, Jones M. The biodegradation of surfactants in the environment. Biochim Biophys Acta. 2000;1508(1-2):235-51. DOI: 10.1016/S0304-4157(00)00013-7

Mixich J, Rothert R, Wullbrandt D. Process for the quantitative purification of glycolipids. United States patent 5656747A. 1997.

Kłosowska-Chomiczewska IE, Mędrzycka K, Hallmann E, Karpenko E, Pokynbroda T, Macierzanka A, et al. Rhamnolipid CMC prediction. J Colloid Interface Sci. 2017;488:10-9. DOI: 10.1016/j.jcis.2016.10.055

Guerra-Santos L, Kappeli O, Fiechter A. Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Appl. Environ Microbiol. 1984;48(2):301-5.

Kuksis A. Chromatography of lipids in biomedical research and clinical diagnosis. 1st ed. Amsterdam: Elsevier; 1987. 459 p.

Seeds of agricultural plants. Methods fоr seed testing. Kyiv: Derzhspozhivstandart Ukrayini; 2003. 148 p. DSTU 4138-2002.

Fire safety. General purpose frothers for extinguishing fires. General technical requirements and test methods. Kyiv: Derzhspozhivstandart Ukrayini; 2016. 32 p. DSTU 3789:2015.

Karpenko O, Banya A, Baranov V, Novikov V, Kołwzan B. Influence of biopreparations on phytoremediation of petroleum-contaminated soil. Pol J Environ Stud. 2015;24(5):2009-15. DOI: 10.15244/pjoes/42672

Karpenko E, Voloshynets V, Karpenko I, Pokynbroda T, Semenyuk I, Midyana H. Сolloidal Characteristics of water systems of rhamnolipid biocomplex of strain Pseudomonas sp. PS-17 with Tween-80 and their prospects for biotechnology. Innov Biosyst Bioeng. 2018;2(1):57-63. DOI: 10.20535/ibb.2018.2.1.127258

Karpenko O, Pokynbroda T, Lubenets V, Martynyuk N, Zin I. Biosynthesis of surfactants by the microorganisms of the genera Pseudomonas in soybean oil and the study of their properties. Visnyk Natsionalnoho Universytetu Lvivska Politekhnika. 2017;868:222-9.

Shulga O, Joegel J, Karpenko O, Prystai M, Vildanova R. Surface-active complexes of the strain Pseudomonas aeruginosa JRV-1. Visnyk Natsionalnoho Universytetu Lvivska Politekhnika. 2017;868:179-86.

SWOT Analysis – Discover New Opportunities, Manage and Eliminate Threats [Internet]. 2019 [cited 2018 Feb 24]. Available from:

Global Biological Surfactant Market 2018 Growth By Players:- Evonik, Agae Technologies, Biotensidon, Ecover, Jeneil Biotech, Logos Technologies - Industry News Today [Internet]. Industry News Today. 2019 [cited 2019 Feb 26]. Available from:




How to Cite

Pokynbroda T, Karpenko I, Midyana H, Karpenko O. Isolation of Surfactants Synthesized by the Pseudomonas Bacteria and Study of Their Properties. Innov Biosyst Bioeng [Internet]. 2019May6 [cited 2024Apr.15];3(2):70-6. Available from: