Mikrobiol. Z. 2018; 80(4):13-27. Ukrainian.
doi: https://doi.org/10.15407/microbiolj80.04.013

Influence of Cultivation Conditions of Rhodococcus erythropolis IMV Ac-5017
on the Properties of Synthesized Surfactants

Pirog T.P.1,2, Shevchuk T.A.2, Petrenko N.M.1, Paliichuk O.I.1, Iutynska G.O.2

1National University of Food Technologies
68 Volodymyrska Str., Kyiv, 01601, Ukraine

2Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

Aim. To establish cultivation conditions of Rhodococcus erythropolis IMV Ac-5017, which provide the synthesis of surfactants with high antimicrobial and antiadhesive activity, as well as high efficiency of oil pollution destruction. Methods. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2 : 1). The number of attached cells and the degree of biofilm destruction were analyzed spectrophotometrically. Antimicrobial properties of the surfactants were determined by index of the minimum inhibitory concentration. The degree of oil destruction was analyzed by its residual concentration, which was determined by the weight method after extraction with hexane. Results. It has been established that cations of calcium are the activator of NADP+-dependent glutamate dehydrogenase (key enzyme of biosynthesis of surfaceactive aminolipids in R. erythropolis IMV Ac-5017). The addition of CaCl2 (0.1 g/l) into cultivation medium of IMV Ac-5017 strain was accompanied by increasing NADP+- dependent glutamate dehydrogenase activity in 2 time and by synthesis of surfactants, the minimum inhibitory concentrations of which with respect to the test cultures were 1.2 − 5 times lower, their adhesion on abiotic materials treated with such surfactants was 12 − 50 % lower, and the degree of biofilms destruction was on average 9 − 10 % higher as compared to indicators for the surfactant produced in the base medium. The introduction of 0.1 mM Cu2+ (activator of alkane hydroxylase - first enzyme of n-alkanes catabolism) in exponential growth phase of IMV Ac-5017 strain on non-hydrocarbon substrates (ethanol, waste oil) was accompanied by formation of surfactants, in the presence of which degree of oil decomposition increased by 8 − 13 % compared with using preparations synthesized in a medium without copper cations. Conclusions. The data presented indicate the possibility of regulating properties of surfactants under producer cultivation. The determining mechanisms underlying this regulation allows development of technologies for production of microbial surfactants, providing synthesis of final product with the necessary predetermined properties, depending on sphere of practical application.

Keywords: Rhodococcus erythropolis IMV Ac-5017, surfactants, antimicrobial and antiadhesive activity, key enzymes, oil degradation.

Full text (PDF, in Ukrainian)

  1. Mnif I, Ghribi D. Review lipopeptides biosurfactants: Mean classes and new insights for industrial, biomedical, and environmental applications. Biopolymers. 2015; 104(3): 129−47. https://doi.org/10.1002/bip.22630
  2. Paulino BN, Pessôa MG, Mano MC, Molina G, Neri-Numa IA, Pastore GM. Current status in biotechnological production and applications of glycolipid biosurfactants. Appl Microbiol Biotechnol. 2016; 100 (24): 10265−93. https://doi.org/10.1007/s00253-016-7980-z
  3. Irorere VU, Tripathi L, Marchant R, McClean S, Banat IM. Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria. Appl Microbiol Biotechnol. 2017; 101(10): 3941−51. https://doi.org/10.1007/s00253-017-8262-0
  4. Fracchia L, Banat JJ, Cavallo M, Ceresa C, Banat IM. Potential therapeutic applications of microbial surface-active compounds. AIMS Bioengineering, 2015; 2(3): 144−62. https://doi.org/10.3934/bioeng.2015.3.144
  5. Franco Marcelino PR, da Silva VL, Rodrigues Philippini R, Von Zuben CJ, Contiero J, Dos Santos JC, da Silva SS. Biosurfactants produced by Scheffersomyces stipitis cultured in sugarcane bagasse hydrolysate as new green larvicides for the control of Aedes aegypti, a vector of neglected tropical diseases. PLoS One. 2017; 12(11): e0187125. https://doi.org/10.1371/journal.pone.0187125
  6. Pidhorskyy V, Iutinska G, Pirog T. [Intensification of microbial synthesis technologies]. Kyiv: Nauk. Dumka, 2010. 327 p. Ukrainian.
  7. Pirog TP, Sidor IV, Lutsai DA. Calcium and magnesium cations influence on antimicrobial and antiadhesive activity of Acinetobacter calcoaceticus IMV B-7241 surfactants. Biotechnologia Acta. 2016; 9(6): 50−7. https://doi.org/10.15407/biotech9.06.050
  8. Pirog TP, Nikituk LV, Shevchuk TA. [Influence of divalent cations on synthesis of Nocardia vaccinii IMV B-7405 surfactants with high antimicrobial and antiadhesion activity]. Mikrobiol Z. 2017; 79(5): 13–22. Ukrainian. https://doi.org/10.15407/microbiolj79.05.013
  9. Singh AK, Rautela R, Cameotra SS. Substrate dependent in vitro antifungal activity of Bacillus sp. strain AR2. Microb Cell Fact. 2014; 13. https://doi.org/10.1186/1475-2859-13-67
  10. Shah V, Badia D, Ratsep P. Sophorolipids having enhanced antibacterial activity. Antimicrob Agents Chemother. 2007; 51(1): 397−400. https://doi.org/10.1128/AAC.01118-06
  11. Das P, Yang XP, Ma LZ. Analysis of biosurfactants from industrially viable Pseudomonas strain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity. Front Microbiol. 2014; 5: 696. https://doi.org/10.3389/fmicb.2014.00696
  12. De Rienzo MA, Martin PJ. Effect of mono and di-rhamnolipids on biofilms pre-formed by Bacillus subtilis BBK006. Curr Microbiol. 2016; 73(2): 183−9. https://doi.org/10.1007/s00284-016-1046-4
  13. Kim K, Lee Y, Ha A, Kim JI, Park AR, Yu NH, Son H, Choi GJ, Park HW, Lee CW, Lee T, Lee YW, Kim JC. Chemosensitization of Fusarium graminearum to chemical fungicides using cyclic lipopeptides produced by Bacillus amyloliquefaciens strain JCK-12. Front Plant Sci. 2017; 8: 2010. https://doi.org/10.3389/fpls.2017.02010
  14. Pirog T, Sofilkanych A, Konon A, Shevchuk T, Ivanov S. Intensification of surfactants' synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium. Food Bioprod Process. 2013; 91(2): 149−157. https://doi.org/10.1016/j.fbp.2013.01.001
  15. Smith EL, Austen BM, Blumenthal KM, Nyc JF. Glutamate dehydrogenases. In: Boyer PD, editor. The Enzymes, 3rd ed., vol. 11. New York: Academic Press; 1975. p. 293−367. https://doi.org/10.1016/S1874-6047(08)60213-9
  16. Bradford M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72 (3): 248–54. https://doi.org/10.1016/0003-2697(76)90527-3
  17. Rivardo F, Turner RJ, Allegrone G, Ceri H, Martinotti MG. Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens. Appl Microbiol Biotechnol. 2009; 83(3): 541–53. https://doi.org/10.1007/s00253-009-1987-7
  18. Kim YG, Kang HK, Kwon KD, Seo CH, Lee HB, Park Y. Antagonistic activities of novel peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum. J Agric Food Chem. 2015; 63(48): 10380−7. https://doi.org/10.1021/acs.jafc.5b04068
  19. Zhang X, Ashby R, Solaiman DK, Uknalis J, Fan X. Inactivation of Salmonella spp. and Listeria spp. by palmitic, stearic, and oleic acid sophorolipids and thiamine dilauryl sulfate. Front Microbiol. 2016; 7: 2076. https://doi.org/10.3389/fmicb.2016.02076
  20. Borsanyiova M, Patil A, Mukherji R, Prabhune A, Bopegamage S. Biological activity of sophorolipids and their possible use as antiviral agents. Folia Microbiol (Praha). 2016; 61(1): 85−9. https://doi.org/10.1007/s12223-015-0413-z
  21. Aleksic I, Petkovic M, Jovanovic M, Milivojevic D, Vasiljevic B, Nikodinovic-Runic J, Senerovic L. Anti-biofilm properties of bacterial di-rhamnolipids and their semi-synthetic amide derivatives. Front Microbiol. 2017; 8: 2454. https://doi.org/10.3389/fmicb.2017.02454
  22. Chong H, Li Q. Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact. 2017; 16(1): 137. https://doi.org/10.1186/s12934-017-0753-2
  23. Wittgens A, Santiago-Schuebel B, Henkel M, Tiso T, Blank LM, Hausmann R, Hofmann D, Wilhelm S, Jaeger KE, Rosenau F. Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida − a step forward to tailor-made rhamnolipids. Appl Microbiol Biotechnol. 2017.
  24. Tiso T, Zauter R, Tulke H, Leuchtle B, Li WJ, Behrens B, Wittgens A, Rosenau F, Hayen H, Blank LM. Designer rhamnolipids by reduction of congener diversity: production and characterization. Microb Cell Fact. 2017; 16(1): 225. https://doi.org/10.1186/s12934-017-0838-y
  25. Wittgens A, Kovacic F, Müller MM, Gerlitzki M, Santiago-Schübel B, Hofmann D, Tiso T, Blank LM, Henkel M, Hausmann R, Syldatk C, Wilhelm S, Rosenau F. Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Appl Microbiol Biotechnol. 2017; 101(7): 2865−78. https://doi.org/10.1007/s00253-016-8041-3
  26. Ribeiro IA, Bronze MR, F Castro M, Ribeiro MH. Selective recovery of acidic and lactonic sophorolipids from culture broths towards the improvement of their therapeutic potential. Bioprocess Biosyst Eng. 2016; 39(12): 1825−37. https://doi.org/10.1007/s00449-016-1657-y
  27. Roelants SL, Ciesielska K, De Maeseneire SL, Moens H, Everaert B, Verweire S, Denon Q, Vanlerberghe B, Van Bogaert IN, Van der Meeren P, Devreese B, Soetaert W. Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola. Biotechnol Bioeng. 2016; 113(3): 550−9. https://doi.org/10.1002/bit.25815
  28. Maddikeri GL, Gogate PR, Pandit AB. Improved synthesis of sophorolipids from waste cooking oil using fed batch approach in the presence of ultrasound. Chem Eng J. 2015; 263: 479−87. https://doi.org/10.1016/j.cej.2014.11.010
  29. Zhao H, Shao D, Jiang C, Shi J, Li Q, Huang Q et al. Biological activity of lipopeptides from Bacillus. Appl Microbiol Biotechnol. 2017; 101(15): 5951−60. https://doi.org/10.1007/s00253-017-8396-0
  30. Ramachandran R, Shrivastava M, Narayanan NN, Thakur RL, Chakrabarti A, Roy U. Evaluation of antifungal efficacy of three new cyclic lipopeptides of the class bacillomycin from Bacillus subtilis RLID 12.1. Antimicrob Agents Chemother. 2018; 62: e01457-17.
  31. Mandal SM, Barbosa AE, Franco OL. Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol Adv. 2013; 31(5): 338−45. https://doi.org/10.1016/j.biotechadv.2013.01.004
  32. Zhihui X, Jiahui S, Bing L, Xin Y, Qirong S, Ruifu Z. Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation. Appl Environ Microbiol. 2013; 79(3): 808−15. https://doi.org/10.1128/AEM.02645-12
  33. Ashby RD, Nu-ez A, Solaiman DKY, Foglia TA. Sophorolipid biosynthesis from a biodiesel co-product stream. JAOCS. 2005; 82(9): 625−30. https://doi.org/10.1007/s11746-005-1120-3
  34. Sleiman JN, Kohlhoff SA, Roblin PM, Wallner S, Gross R, Hammerschlag MR, Zenilman ME, Bluth MH. Sophorolipids as antibacterial agents. Ann Clin Lab Sci. 2009; 39(1): 60−3.
  35. Pirog TP, Konon AD, Sofylkanych AP, Shevchuk TA, Parfenyuk SA. [Effect of Cu2+ on synthesis of biosurfactants of Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Ac-5017]. Mikrobiol Z. 2012; 75(1): 3–13. Russian.
  36. Pirog TP, Konon AD, Sofilkanich AP, Shevchuk TA, Iutinska GO. [Destruction of oil in the presence of Cu2+ and surfactants of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405]. Mikrobiol Z. 2015; 77(2): 2–8. Russian. https://doi.org/10.15407/microbiolj77.02.002
  37. Ławniczak Ł, Marecik R, Chrzanowski Ł. Contributions of biosurfactants to natural or induced bioremediation. Appl Microbiol Biotechnol. 2013; 97(6): 2327–39. https://doi.org/10.1007/s00253-013-4740-1