Langmuir 1993,9, 1634-1636
1634
Colloid Chemical Effect of Polar Head Moieties of a Rhamnolipid-Type Biosurfactant Yutaka Ishigami,'*t Yasuo Gama? Fumiyoshi Ishii: and Young Kook Choir National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305, Japan, Meiji College of Pharmacy, Tanashi, Tokyo 188, Japan, and Korea Research Institute of Chemical Technology, Daedeog-Danji, Daejeon 305-606, Korea Received August 17,1992. I n Final Form: March 29, 1993 Rhamnolipid B (RB), a microbiallyexcreted biosurfadant,was characterized by interfacialand biological activities in comparison with the corresponding methyl ester (RB-Me) and divalent cationic salts. RB may be able to control interfacial behavior by the functional conversion of carboxylic moieties depending on the environmental pH, as RB has two hydrophilic moieties of diaaccharide and carboxylic acid. On the other hand, RB-Me showed a large wetting action for polymer surfaces and biomembranes, and a considerable lowering of the interfacial tension (0.1 mN/m) at the octane-solution interface. cowhide for skin, and silkworm cocoon, respectively.- Rat abdominal skin was prepared from a male hairless rat (180-g Rhamnolipid B, as well as rhamnolipid A, the precursor weight). Measurements. The contact angle was measured using a of rhamnolipid B, has been known to be produced as a goniometer (Eruma Optical Co. M-2010A). The surface tension biosurfactant by hydrocarbon-assimilable procaryote8 in was measured using a Wilhelmy-typesurface tensiometer (Shiorder to take hydrocarbon droplets inside their cell madzu ST-1). Interfacial tension was measured usinga spinningmembranes.lP2 Rhamnolipid A and B show excellent droptype interfacial tensiometer (Core Laboratories Co. Model surface activities in spite of their bulky and complicated 500). The determination of the critical micelle concentration structure^.^^^ From this point of view, we attempted to (cmc) was carried out by 8-anilinonaphthaleneeulfonate(ANS) elucidate the precise biological system relating to the fluorophotometryusing a fluorophotometer (ShimadzuRF-640). Hemolytic actions of RB and RB-Me were measured at 543 nm amphipathic function. in ISOTON buffer (pH 7.2) isotonic to human blood. Rabbit (2 mL) were mixed with sample solutions (2 mL) erythrocytes Materials and Methods and incubated at 30 O C for 30 min. The 100% hemolysis level Rhamnolipid B ([[[2-0(2-Ou-Decenoyl-u-~-rhamnopy- was defied as the absorbancy of hemoglobin after the complete ranosyl)-u-~-rhamnopyranosyl]-3-decanoyl]o~]-3-d~an~hemolysis of erythrocytes in distilled water. ic Acid), Rhamnolipid B (RB)was isolated from the culture Results and Discussion broth of Pseudomonas BOP 100 cultivated in inorganic media RB was containing 0.1 9% hexadwane as the sole carbon sourc~.~ (1) Comparison of Surface Activities of RB and purified by preparative HPLC (Waters Co.) (BondasphereCleRB-Me. It is difficult to understand the amphipathic 100 column; flow rate, 10 mL/min, eluent, acetonitrile/O.Ol M behavior of RB molecules, because RB has two hydroKzHPOr (46;pH 6.6)). phobic ((3-decanoyloxy)-3-decanoyland decenoyl)and two Rhamnolipid B Methyl Ester (RB-Me) and Its Metal hydrophilic (dirhamnosyl and carboxyl)moieties,as shown Salts. RB-Me was prepared by the reaction of RB with methyl in Figure 1. It was attempted to compare RB with RBiodide in the presence of 1,8-diazabicycl0[5.4.0]-7-undecenein Me in the presence and the absence of the RB carboxylic benzene and was isolated by column chromatography on silica moiety, as the chemical structure of a,u-double hydrophilic gel: colorlegs oil; IR 1738cm-' (COOCH& 'H NMR (CDCb) 6 moieties of dirhamnosyl and carboxylicgroups is not usual 3.68 (8, 3H, COOCHa). Mg, Ca, and Ba salta of RB by exchanging in comparison with synthetic surfactants. T h e pHgegenion species of RB-Na using MgC12, CaC4, and BaClz dependent conversion of surface activities may play an solutionswere recrystallized from ethanol-water: IR (COO-) RBimportant role in the growth of the hydrocarbon-assimNa (1568cm-l), RB-Mg (1567cm-'), RB-Ca (1543cm-l), RB-Ba ilable Pseudomonas which produce RB. In fact, RB is (1661cm-1). SDS (sodium dodecyl sulfate) was supplied by Kao Corp. neutralized completely at pH 7.8 with NaOH, and the optimum growing pH is around 7.3. Comparative results Membrane. Polymer plates were purchased from Nippon of the surface activities of RB and RB-Me are summarized Test Panel Co. The plates have proper surface characteristics for hydrophilicity6 of Teflon (Ten, 18.6; polyethylene (PE), 31; in Table I. The cmc values of RB-Na and the correpolystyrene (Pst), 33; poly(methy1methacrylate) (PMMA),39; sponding surface tensions (ycmc) are smaller than those of and poly(ethy1eneterephthalate) (PET),43 mN/m. The keratin, RB-Me, while aqueous RB-Na solution shows a large collagen, and fibroin membraneswere prepared from hen feather, interfacial tension (3.6 mN/m) in comparison with RBMe (0.1 mN/m). The former result s e e m to reflect the * To whom correspondence should be addressed. high efficiency of RB. Both values are sufficiently low to + National Chemical Laboratory for Industry. realize the emulsification of hydrocarbon droplets cont Meiji College of Pharmacy. cerning their interfacial tension lowering. It was found 5 Korea Reeearch Institute of Chemical Technology. that Serratia species form their exclusivegrowing colonies (1)Yamaguchi, M.; Sato, A.; Yukuyama, A. Chem. I d . 1976,4,741. (2) Yamaguchi, M.; Sato, A.; Dazai, M.; Takahara, Y. Rep. Ferment. Res. Inst. 1978, No.51,51. (6) Sugiura,M.;Kikkawa,M. Bull. Gou. Chem.Res. Inst. (TokyoKogyo (3) Iahigami, Y.; Gama, Y.; Nagahora, H.; Yamaguchi,M.; Nakahara, Shikemho Hokoku) 1977, 72,l. H.;mta,T. Chem. Lett. 1987, 763. (7) Sugiura,M.; Shinbo, T.; Kikkawa, M. Bull. Gou. Chem. Res. Zmt.
Introduction
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(4) Iehigami, Y .;Gama, Y.; Yamaguchi, M.; Nakabara,H.; Kamata,T. J. Jpn. Oil Chem. SOC.(Yukagaku) 1987,36, 763. (5)Wu, S.J. Phys. Chem. 1968, 72,3332.
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(Tokyo Kogyo Shikemho Hokoku) 1974,69, 274. (8) Sugiura, M.Bull. Gou. Chem. Res. Inst. (Tokyo Kogyo Shikensho Hokoku) 1974,69, 54.
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Rhamnolipid- Type Biosurfactant
Langmuir, Vol. 9, No.7,1993 1635
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Figure 2. Time course of interfacial tension between the drop of CHC4 and the bulk aqueous solution of RB salts at 30 "C.
CPK model of rhamnolipid B.
Figure 1.
Y
120 180 Time (min)
60
Table I. Surface Activities of Rhamnolipid Homologues rhamnolipid B (RB-Na)
methyl ester of rhamnolipid B (RB-Me)
~~
28.0 mN/m 30.6 mN/m rcmc 3.5 mN/m (0.1%) 0.1 mN/m (0.01%) interfacial tension for octane 3.15 X 1V M (0.026%) 4.95 X 10-4 M (0.040%) cmc (ANS fluorometry)
in low interfacial tension territories by excreting biosurf a ~ t a n t .In ~ addition, the attainment of lower interfacial tension valueslO by RB-Me may be harmful for the excreting cell bodies in the environment. The cell thus does not excrete RB-Me. We attempted to elucidate the effects of RB-Na on chloroform droplets as a model cell surface, because chloroform has a critical surface tension of 27.1 mN/m (20 "C) as with hydrocarbon-assimilable cell sUrfaces.ll Aqueous RB-Na solution (0.1%) has an interfacial tension of 0.3 mN/m (1min) for chloroform drops (ca. 3 pL), and the values decreased sharply until the chloroform drop disappeared at 6 min, as shown in Figure 2. The disappearence of chloroform means the attainment of interfacial tension corresponding to zero. Free acid type RB was known to form vesiclesitself4as a self-organizingassembly in the pH range of 4.3-5.8 (pKa 5.6). Furthermore, RB can catch the surrounding alkaline-earth metals in the weakly acidic pH of the environment. The resulting Mg, Ca, and Ba (reference of artificial gegenion) salts of RB are able to control the surface tension at the air-water interface, as shown in Figure 3. It was reported that microbial growth is enhanced in the media of the surface tension region of 45-60 mN/m and of the corresponding concentrations of O.Ol-O.l% in the case of saponin.1° Therefore, it is noteworthy that RB-Na has the lowest ycmc value and divalent Ba salt has the lowest cmc values of the four kinds of metal salts. (2) Lipophilic Properties of the Cell Surfaces of Hydrocarbon-AssimilableMicroorganismsandTheir Affinity for Artificial and Intact Biomembranes. Various kinds of solid surfaces have a proper hydrophilic-lipophilic balance (HLB). It is known that the ~~~
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(9) Matsuyama, T.;Sogawa, M.;Nakagawa, M. FEMS Microbiol Lett. 1989,61, 243. (10) Kanie, T. PPM 17 (lo), 28. (11)Gerson, D. F.; Zajic, J. F. Immobilized Microbial Cells; ACS
Symposium Series; Washington, DC, 1980, Vol. 106, p 29.
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Figure 3. Surface tension vs concentration plots of RB metal salts at 30 "C.
RB-Me __ O- 0.10% R, -N B-a --.
